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The 7kDa Sso7 is a basic protein particularly abundant in Sulfolobus solfataricus and is involved in DNA assembly. This protein undergoes in vitro ADP-ribosylation by an endogenous poly(ADP-ribose) polymerase-like enzyme. The circular dichroism spectrum of purified ADP-ribosylated Sso7 shows that this modification stabilizes the prevalent protein β-conformation, as suggested by shifting of negative ellipticity minimum to 220nm. Moreover, a short ADP-ribose chain (up to 6-mers) bound to Sso7 is able to reduce drastically the thermoprotective and DNA condensing ability of the protein, suggesting a possible regulatory role of ADP-ribosylation in sulfolobal DNA organization.

The partial amino acid sequence of the sulfolobal thermoprotein biochemically characterized as poly(ADP-ribose)polymerase-like enzyme overlaps those of DING proteins. This group of proteins, widely occurring in animals, plants and eubacteria, shows a characteristic and highly conserved N-terminus, DINGGGATL. The sequence of the N-terminal region and of the analyzed tryptic peptides of the sulfolobal thermozyme shows a high similarity with most of the DING proteins from databases. This is the first example of a DING protein from a sulfolobal source.

A chromatin fraction, named pP fraction, was prepared from rat testis nuclei, which had been digested with nuclease in order to separate soluble and insoluble chromatin. This fraction resembled nuclear matrix as it was highly resistant to DNAase digestion, had a high content of proteins compared to the low DNA percentage, and a noticeable transcriptional activity. Moreover, poly(ADPribosyl)ation system (i.e., poly(ADPR)polymerase, poly(ADPribose), and acceptor proteins) was still present at high levels. In order to study whether it might be identified as the protein support surrounding chromatin loops, this pP fraction was further analyzed after 3 M NaCl extraction. The 3 M NaCl extract and the highly insoluble pellet, named Nuclear Matrix Pellet, were characterized as it regards DNA, newly synthesized RNA and proteins. Furthermore, poly(ADPribose) metabolism was analyzed by measuring both poly(ADPribose) polymerase and poly(ADPribose) glycohydrolase activities, poly(ADPribose) distribution and by identifying protein acceptors. The final pellet had features of nuclear matrix containing less than 10% DNA and high percentage of proteins; 28% of newly synthesized RNA was still associated with this fraction. Long and branched polyADPribose were found in the nuclear matrix-like pellet, although ADPribose acceptors (mainly H1 and core histones) appeared to be modified mostly with short ADPribose oligomers. Longest and branched polymers were retained on the top of protein gel, likely bound to automodified poly(ADPribose) polymerase. J. Cell. Biochem. 89: 688–697, 2003. © 2003 Wiley-Liss, Inc.

The poly(ADPribose) polymerase-like thermozyme from the hyperthermophilic archaeon S. solfataricus was found to bind DNA with high affinity and non-specifically. Binding was independent of base composition and length of the nucleic acid, and the protein showed a slight preference for the circular structure. By using pCMV-Neo-Bam plasmid as experimental model, the behaviour of the thermozyme upon binding with either circular or linear plasmid was analyzed. pCMV-Neo-Bam has a single HindIII site that allows to obtain the linear structure after digestion with the restriction enzyme. Intrinsic tryptophan-dependent fluorescence of poly(ADPribose) polymerase-like thermozyme noticeably changed upon addition of either circular or linear plasmid, showing the same binding affinity (K=2 x 10(9) M-1). However, experiments of protection against temperature and DNase I gave evidence that the thermozyme formed more stable complexes with the circular structure than with the linear pCMV-Neo-Bam. Increasing temperature at various DNA/protein ratios had a double effect to reduce the amount of circular DNA undergoing denaturation and to split the melting point towards higher temperatures. Nil or irrelevant effect was observed with the linear form. Similarly, DNase acted preferentially on the linear plasmid/protein complexes, producing an extensive digestion even at high protein/DNA ratios, whereas the circular plasmid was protected by the thermozyme in a dose-dependent manner. The complexes formed by archaeal poly(ADPribose) polymerase (PARPss) with the circular plasmid were visualized by bandshift experiments both with ethidium bromide staining and by labelling the circular plasmid with 32P. The stability of complexes was tested as a function of enzyme concentration and in the presence of a cold competitor and of 0.1% SDS. From the performed experiments, a number of 3-10 base pairs bound per molecule of enzyme was calculated, indicating a high frequency of binding. The presence of circular DNA was also able to increase by 80% the poly(ADPribose)polymerase-like activity, as compared to 25% activation induced by the linear pCMV-Neo-Bam.

Rat testis H1 proteins were poly(ADP-ribosyl)ated in vitro. The modifying product, poly(ADP-ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis-specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP-ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP-ribose)polymerase in the presence of [(32)P] NAD. In parallel, poly(ADP-ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [(32)P] NAD. In both experiments, the poly(ADP-ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP-ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10-12 units long, whereas longer chains (

A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose. The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold. The molecular mass (54-55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was proved to be thermophilic, with a temperature optimum of approx. 80 degreesC, and thermostable, with a half-life of 204 min at 80 degreesC, in good agreement with the requirements of a thermozyme. It displayed a Km towards NAD+ of 154+/-50 microM; in the pH range 6.5-10.0 the activity values were similar, not showing a real optimum pH. The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay. The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source. Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose). These results strongly suggest that the activities of the purified enzyme include the elongation step.

The rat testis chromatin fractions (soluble, S, and insoluble, P) were prepared by mild digestion of nuclei with DNAase I. They appeared to be different in specific biochemical features such as their transcriptional competence and protein patterns, the latter indicating according to results previously obtained, that the testis-specific H1t is preferentially associated to the soluble fraction, whereas the other H1 variants are localized in the pellet. S and P chromatins also differed in the distribution of the poly(ADP-ribosyl)ating system, (poly(ADP-ribose)polymerase, reaction product and acceptor proteins), detected by incubating nuclei with 32P-NAD. The 32P-modified H1s and core histones of both fractions, known as specific ADPribose target proteins, were separated by high performance liquid chromatography and it was demonstrated that the H1 variants from S and P are differently ADPribosylated, being H1t always the best acceptor, and that most of the ADPribosylated variants were solubilized after DNase I treatment. The further digestion of P chromatin with the nuclease produced a fraction (pP) devoid of most DNA, but particularly enriched in transcriptionally competent tracts. The low DNA content of pP chromatin, which reflects the typical feature of a nuclear matrix, corresponded to a relevant poly(ADPribosyl)ation, the highest as compared to S and P fractions. Moreover, long and branched chains of poly(ADP-ribose) were found associated to pP sample which resemble the products determined in the soluble chromatin.

Due the likely role of H1 histone variants in inducing the formation of folded DNA filaments with different stabilities, the condensing capacity of the testis-specific H1t versus the somatic variants was tested. Circular dichroism analyses of rat testis H1-depleted oligonucleosomes (5-2kbp) revealed that H1t, which appears in germ cells during the meiotic prophase of mammalian spermatogenesis, exerts the lowest condensing effect as compared to the other variants. The distribution of H1 subtypes among different chromatin fractions was also investigated and gave evidence that H1t is more abundant in chromatin regions which are more sensitive to DNAase I digestion.

Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has increased in the last decade. Recent advances in the understanding of the underlying biology of the progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-kappaB (NF-kappaB) pathway. In vitro studies have shown that NF-kappaB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. It has been found that a short cell-permeable peptide spanning the IKK-beta NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFalpha-induced NF-kappaB activation in vivo. In the present study we investigated the effect of the NBD peptide on NF-kappaB activity and survival of A375 human melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-kappaB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-kappaB DNA-binding activity and induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process.

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

The controversy about the occurrence of an (ADPribosyl)ating activity in yeast is still standing up. Here we discuss this topic on the basis of results obtained with classic experiments proposed over years as basis to characterize an (ADPribosyl)ation system in any organism. Independent results obtained in two different laboratories were in line with each other and went towards the occurrence of an active (ADPribosyl)ating system in Saccharomyces cerevisiae. In fact data collected from nuclear preparations of cultured cells matched those from baker's yeast and lyophilized yeast cells. Yeast (ADPribosyl)ating enzyme is a protein of 80–90 kDa, as determined by electrophoresis on polyacrylamide gel in sodium dodecyl sulphate, followed by immunoblotting with antibodies against anti-poly(ADPribose) polymerase catalytic site. It synthesizes products, that, after digestion with phosphodiesterase, co-migrates mainly with phosphoribosyl adenosine monophosphate after thin layer chromatography on silica gel plate. © 2005 Wiley-Liss, Inc.

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.

In this study, we have identified a 28-kDa protein resembling the linker H1 in the testis and prostate of the reproductive system of Octopus vulgaris. This protein, OvH1, was partially purified by reverse phase high-pressure liquid chromatography (HPLC) of the perchloric acid extract from testis nuclei. It showed electrophoretic mobility, CD spectrum and amino acid composition highly comparable with those of the mammalian histone. Moreover, it was microheterogeneous, as resulted from prostate and testis HPLC and mass spectrometry analyses. Such analysis showed that in testis there are two H1 subfractions, which do not appear in the prostate. Amino acid composition of the major testis specific variant (OvH1t) showed high similarity with rat testis specific H1t. The histone-like nature of OvH1 was confirmed by its ability to bind DNA as tested both by circular dichroism and protection of the nucleic acid toward deoxyribonuclease I activity. The circular dichroism spectra of Octopus DNA in the absence and presence of increasing amounts of the protein showed a dose-dependent effect, leading to a progressive compactness of the polynucleotide. OvH1/DNA complexes were also resistant to nuclease digestion. The presence of H1 in the testis and prostate of the reproductive system of Octopus is discussed in light of the fact that there is a similarity between its behavior and that of vertebrates. Mol. Reprod. Dev. 63: 355–365, 2002. © 2002 Wiley-Liss, Inc.

The ADPribosylating enzyme from the thermophilic archaeon S. solfataricus was purified by a simple procedure which included preparative electrophoresis on a 0.1% SDS- polyacrylamide gel. The gel slice containing the enzymatic protein was cut out and the enzyme was solubilized by electroelution. The pure enzyme was obtained by chromatography of the electroeluted sample on a DNA-Sepharose column. The purified enzyme retained both its full activity and the structuring ability as a function of temperature increase.

The amino acid sequence of a novel tissueand phasespecific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 ± 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the Cterminus. They have thus been named [des(Ala[81]) SNP1] and [des(Lys[80]Ala[81]) SNP2], respectively. The molecular weights are 9140.21 ± 0.83 for [des(Ala[81]) SNP1] and 9011 ± 0.09 for [des(Lys[80]Ala[81]) SNP2].

Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradation of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.

In the archaeon Sulfolobus solfataricus, protein ADPribosylation by free ADPribose was demonstrated by testing both [adenine-14C(U)]ADPR and [adenine- 14C(U)]NAD as substrates. The occurrence of this process was shown by using specific experimental conditions. Increasing the incubation time and lowering the pH of the reaction mixture enhanced the protein glycation by free ADPribose. At pH 7.5 and 10 min incubation, the incorporation of free ADPribose into proteins was highly reduced. Under these conditions, the autoradiographic pattern showed that, among the targets of ADPribose electrophoresed after incubation with 32P-NAD, the proteins modified by free 32P-ADPribose mostly corresponded to high molecular mass components. Among the compounds known to inhibit the eukaryotic poly-ADPribose polymerase, only ZnCl2 highly reduced the ADPribose incorporation from NAD into the ammonium sulphate precipitate. A 20% inhibition was measured in the presence of nicotinamide or 3-aminobenzamide. No inhibition was observed replacing NAD with ADPR as substrate. J. Cell. Biochem. 66: 37–42, 1997. © 1997 Wiley-Liss, Inc.

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).

Poly(ADPribosylation) of nuclear proteins has been investigated in the nuclei from growing oviducts of intact and estrogen-treated spayed females of the lizard Podarcis s. sicula Raf. Isolated nuclei were incubated with [14C]NAD and nuclear proteins extracted in 0.2 M H2SO4. Labeled acid-soluble proteins were analysed by reverse-phase high-performance liquid chromatography (HPLC) and acetic acid-urea gel electrophoresis. The results reported here indicate that the ADPribosylation reaction is involved in modifying, besides histone H2b, tissue specific proteins (SNPs and LMG-O). Moreover, comparable results have been obtained from nuclei prepared from the fully active oviduct of intact animals and spayed lizards stimulated with 17β-estradiol. It is concluded that the poly-(ADPribosylation) of hormone-induced proteins might play a role in the differentiation of the lizard oviduct.

The nuclear acceptor proteins for poly(ADP-ribose) were investigated in mouse liver and testis. In liver, histones are ribosylated preferentially, whereas in testis the major acceptors are non-histone proteins. An analysis of the purified testicular acceptor proteins suggests that they are high- and low-mobility-group-like proteins.

ADP-ribosylation of a specific basic protein has been investigated in isolated intact bull testis nuclei incubated with NAD + . The electrophoretic mobility, molecular weight and amino-acid composition of the purified bull testis specific protein are similar to those of rat testis protein. About 1–5% of the total radioactivity incorporated in the 20% acid-insoluble fraction was associated with testis protein and was identified as ADP-ribose.

Polyclonal antibodies raised against eukaryotic mono(ADPribose)transferase and poly(ADPribose)polymerase were used to test the presence of antigenic determinants in a crude extract of Sulfolobus solfataricus, a thermophilic archaeon. Samples from eukaryotic (bull testis) and bacterial (E. coli) sources were analysed for comparison. All tested antibodies reacted with the sulfolobal sample with a specificity comparable to that of the eukaryotic preparation, as revealed by ELISA test, activity assays in the presence of antibodies and immunoblot experiments. After electrophoresis and western blot of sulfolobal proteins, a band at a mass around 50 kDa was detected by immunostaining.

The activity of purified bovine seminal RNAase and pancreatic RNAase A (EC 3.1.27.5) has been investigated following in vitro ADPribosylation in the presence of nuclear ADPribosyltransferase (EC 2.4.2.30) and NAD + . ADPribosylation of these enzymes was correlated with a significant decrease in their activities. Approximately three residues of ADPribose were present per mol of enzyme. Removal of the bound ADPribose restored enzyme activity to near normal levels. Similar results were obtained with nuclei isolated from bull seminal vesicles as an endogenous source of seminal RNAase and nuclear ADPribosyltransferase. The findings suggest that in vitro ADPribosylation has a reversible inactivating effect on ribonucleases.

Covalent modification of enzymes can be effected by ADP-ribosylation reactions. Examples in this regard are represented, in prokaryotes, by elongation factor 2 (EF-2) of protein synthesis, a peptidyl transferase that is inhibited when acted upon in the presence of NAD+ by diphteria toxin, which is endowed with pApase activity (Honjo et al. 1971); as a result, protein synthesis is blocked. A similar mechanism operates in the infection by Pseudomonas aeruginosa, the toxin of which also promotes ADP-ribosylation of EF-2 (Iglewski and Kabat 1975). Also T4 phage-infected E. coli RNA-polymerase is modified, and thereby inhibited, by ADP-ribosylation at arginine residues of the α subunits (Goff 1974). A modification of the adenylate cyclase system has been recently found in a number of instances; it would appear that ADP-ribosylation of the GTP-binding protein component of that system leads to reduction of GTP hydrolysis, and thereby to enhanced adenylate cyclase activity (Gill et al. 1979). In eukaryotes, a Ca++, Mg++-dependent endonuclease of rat liver nuclei has been reported by Yoshihara et al. (1975) to be ADP-ribosylated by an enzyme preparation from the same tissue.

Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA.

The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.

Poly(ADP-ribosylation) of nuclear proteins is one of a number of post-translational events which has been demonstrated in eukaryotic cells. The reaction is catalysed by poly(ADP-ribose) synthetase which transfers ADP-ribose from NAD to a suitable acceptor protein. Histone H1 and HMG proteins 1, 2, 14 and 17 have been reported as acceptors in a variety of tissues ranging from trout testis [1] to mammary carcinoma cells [2]. Poly(ADP-ribosylation) of HMG proteins is of particular interest because of the reported association of these proteins with actively transcribed genes [3]. Functionally, poly(ADP-ribosylation) has been implicated in a variety of regulatory events such as DNA synthesis [4], DNA excision repair [5, 1], gene expression [6] and cell differentiation [7].

The thermophilic microorganism Sulfolobus solfataricus is able to grow at low pH (3.5) and high temperature (87°C) and has been isolated from an acidic hot spring in Agnano (Napoli), Italy (1). This bacterium belongs to the archaebacteria, a phylogenetic group of microorganisms that can be distinguished from other bacteria and eukaryotes (2, 3). The archaebacteria group was initially defined on the basis of a partial 16S ribosomal RNA sequence comparison, which has been well supported by other biochemical features (2-4). More recent studies by Woese and collaborators, based on comparison of complete 16S ribosomal RNA sequences, confirmed and extended earlier archaebacteria phylogeny (3). Although archaebacteria taxonomy is complex and controversial, three main phenotypes have been described, characterized by their peculiar ecological niches, namely high salt environments (halophilic and haloalkaliphilic archaebacteria), high temperature environments (thermophilic and sulfur-dependent archaebacteria) and stringent anaerobic conditions (methanogenic archaebacteria). Several features indicate that the sulfur-dependent branch of archaebacteria, to which Sulfolobus solfataricus belongs, is more closely related to eukaryotes than eubacteria or other archaebacteria (3). One example is the diphtheria toxin reaction, which catalyzes the transfer of ADP-ribose to eukaryotic elongation factor 2. Kessel and Klink (5) demonstrated that the toxin is able to catalyze, in several archaebacteria including Sulfolobus solfataricus, the covalent binding of ADP-ribose to elongation factor, EF-2.

The effects of analogs of diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) were examined on the ADP-ribosylation reaction of histone H1 catalysed by purified bovine thymus poly(ADP-ribose)transferase. Among the compounds tested, epsilon Ap4A and ApCH2pppA were shown to be the most efficient inhibitors of the enzyme. From kinetic studies of their action, it appears that epsilon Ap4A and ApCH2pppA might be 'mixed type' inhibitors.

Many experimental data have been obtained regarding the physiological functions of poly(ADP-ribosyl)ation reactions. DNA replication, cell differentiation, transformation of cells, and DNA repair are among the processes in which poly(ADP-ribosyl)-ation is thought to be involved [1–3]. Inhibitors of ADP-ribosyltransferase (ADPRT) have been very useful in the attempt to ascribe a physiological role to these reactions in various cellular functions. 3-Aminobenzamide and 3-methoxybenzamide have been shown to be powerful competitive inhibitors of ADPRT [4]. Some experimental data obtained with the use of these inhibitors clearly indicate that poly(ADP-ribosyl)ation reactions are indeed crucial for cell differentiation [3] and the DNA strand break rejoining process [5]. On the other hand, the use of inhibitors often meets inconveniences in that their effects are not always limited to the desired metabolic pathway [6]. Tanaka et al. [7] have recently reported that diadenosine tetraphosphate, a ligand of a subunit of DNA polymerase α, could act as a very efficient inhibitor of ADPRT, but further data concerning its involvement in ADP-ribosylation reactions have not been reported so far. Poly amines, such as spermine and spermidine, are also known to have inhibitory effects on ADP-ribosylation of histone H1 [8], although their usefulness as a specific inhibitor of ADPRT remains doubtful. In the light of this circumstance, we have tried to examine the effect of various 2′,5′-oligoadenylates (2′,5′A) on the activity of ADPRT.

The enzyme poly(adenosine diphosphate ribose)polymerase was discovered in chicken liver nuclei in 1966 (Chambon et al., 1) and thoroughly studied in a number of laboratories, where its main properties have been investigated; major contributions in this regard have been given by the groups of Hayaishi (2) and Sugimura (3). The enzyme has many peculiar properties.

ABSTR. 62

Summary When native RNAase BS-1 x is treated with cyanogen bromide, one peptide (CB2) is cleaved from the rest of the protein (CB1) and can be isolated by gel filtration. Peptide CB2 has been sequenced and shown to be the N-terminal peptide of the protein, strictly homologous to the peptide 1–13 of RNAase A. A second fragment (peptide PF2) can be separated by gel filtration after performic acid oxidation of the CB1 fragment. The sequence of this peptide is partially homologous to the 14–29 sequence of RNAase A. From the recovery values determined for peptides CB2 and PF2, it appears that each peptide is present in both chains of RNAase BS-l. These and previous findings are briefly discussed in terms of structure to function relationships.

We have previously demonstrated that a significant percentage of poly(ADPR) polymerase is present, as a tightly-bound form, at the third level of chromatin organisation defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [14C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix.