Marie-Elisabeth Ittel

Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization.

The interaction between purified calf thymus poly(ADP‐ribose) polymerase and its activating co‐purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme‐DNA complex possesses a nucleosome‐like structure. The enzyme‐bound DNA (sDNA) was found to be enriched in single‐stranded regions and branched structures, presumed to be replication forks. The auto‐ribosylated polymerase as well as the branched poly(ADP‐ribose) formed were visualized by dark field electron microscopy during the auto‐ADP‐ribosylation reaction and the possible mechanism of this phenomenon is discussed.

Involvement of polyadp-ribose polymerase in the initiation of phytohemagglutinin induced human lymphocyte proliferation

Nicotinamide (10 mM) or 3-aminobenzamide (5 mM) added at the onset of phytohemagglutinin (PHA) treated human lymphocyte cultures provoke a marked inhibition of the PHA induced DNA synthesis and cell proliferation as well as of poly(ADPR) polymerase activity. When the inhibitors of poly(ADPR) polymerase are added at a later stage of culture (48 h) no inhibition of the stimulation of DNA synthesis and cell proliferation by PHA in human lymphocyte cultures is observed. The intervention of ADP ribosylation at the initiation of DNA synthesis is suggested.

The presence of transthyretin in rat ependymal cells is due to endocytosis and not synthesis

The presence and synthesis of transthyretin, a major carrier protein of thyroxine in rat cerebrospinal fluid, was investigated in choroid plexus epithelial cells and ependymal cells by immunocytochemistry, in situ hybridization, and analysis by Northern and Western blot using a specific oligonucleotide probe and a specific polyclonal antibody to transthyretin. Choroid plexus epithelial cells expressed transthyretin at high levels in developing rat cerebral hemispheres and in cultured cells. These cells secreted transthyretin into the cerebrospinal fluid. In the developing rat brain transthyretin was present in the cytoplasm of ependymal cells, in vesicles in contact with the apical membrane and in cilia. In ependymal cell cultures this protein was particularly abundant in the cilia of these cells. In contrast, ependymal cells did not synthesize transthyretin. It is postulated that transthyretin is transported to ependymal cells from the cerebrospinal fluid by endocytosis.

Poly(adenosine diphosphate ribose) polymerase activity in neuronal and glial nuclei from bovine cerebrum.

Two different preparations isolated from beef cerebrum have been used to compare the polyadenosine diphosphate ribose (polyADPR) polymerase activities in neuronal and glial nuclei: (1) nuclear suspensions (with or without DNase I treatment), and (2) 1 M NaCl nuclear extracts (soluble enzyme). The DNAse I treatment of nuclei and the solubilization of polyADPR polymerase by 1 M NaCl enhances the polyADPR polymerase activity. The polyADPR polymerase activity is similar in neuronal and glial nuclear suspensions, while the neuronal soluble enzyme activity is significantly higher than that of the glial soluble enzyme. Evidence is presented that the difference in soluble enzyme activities is not due to the effects of DNA or degrading enzymes. Some activating factor(s) seem to be present in neuronal soluble extracts, while both inhibiting and activating factor(s) seem to be present in glial soluble extracts.

Similarities in the molecular weight of poly(ADPR) polymerase from different tissues and species

Abstract Using the protein blotting technique, we determine the molecular weight of polyADPR polymerase from different tissues of beef, rat, chicken and pig directly in nuclear homogenates. We report here that the molecular weight of the enzyme is 130,000 in all samples tested.

Kinetics of Nucleosomal Histone H1 Hyper(ADP-Ribosylation) and Polynucleosomes Relaxation

Poly(ADP-ribose) polymerase, a chromatin-bound enzyme, catalyzes postsynthetic modifications of various nuclear proteins through the covalent attachment of ADP-ribose units at the expense of the cellular NAD pool [1–3]. Poly(ADP-ribosylation) appears to be involved in DNA excision repair, cellular proliferation, and differentiation [1–3] and has been shown to induce architectural changes in chromatin [4–6].

Polyadp-Ribose Polymerase and ADP-Ribosylation Reaction

Twenty years ago, polyADP-ribose, poly(ADPR), and poly(ADPR) polymerase, poly(ADPR)-P, were discovered in liver nuclei. A growing body of evidence suggests that poly(ADPR)-P, which appears to also be a poly(ADPR) transferase, is ubiquitously distributed in eukaryotic nuclei and involved in DNA replication, transcription and repair. Poly(ADPR) or mono(ADPR) transferase reaction is present in several subcellular fractions, including mRNP particles. Poly(ADPR)-P activity is very high in neurons. Rather lower activity exists in normal and transformed astrocytes. Nicotinamide (Nic) or 3-aminobenzamide (Nic analogue), known poly(ADPR)-P inhibitors, decrease proliferation of normal astrocytes and C6 glioma cells, as reported for other proliferating cell types.

Sequence Analysis of the DNA Associated with Poly(ADP-Ribose) Polymerase Molecules

Since the first description of an eukaryotic ADP-ribosylation system it has been shown that DNA is required for the synthesis of the polymer (1, 2). During the past several years, the enzyme has been purified to homogeneity from various sources (3, 4). It has been demonstrated that the purified enzyme absolutely requires double stranded DNA to express its activity in vitro (5–8), nevertheless covalently closed circular double-stranded DNA does not have any effect on the enzyme activity (9, 10). It has been shown that poly(ADP-ribose) polymerase is activated by and binds to, single or double stranded breaks on DNA (10).

Chicken poly(ADP-ribose) polymerase. Complete protein sequence deduced from cDNA,comparison with mammalian enzyme sequences

In the past few years human poly(ADP-ribose)polymerase (pADPRP) cDNA (Uchida et al., 1987, Kurosaki et al., 1987) as well as the bovine (Saito et al., 1990), mouse (Huppi et al., 1989) and part of the rat (Thibodeau et al., 1989) cDNAs have been cloned revealing an extensive aa sequence conservation in mammals. To further study the interspecies conservation, we isolated a 1.8 kb human pADPRP cDNA clone from an oligo (dT) primed lgt10 cDNA library (Walter et al., 1985). This clone encodes the entire automodification domain of the enzyme and a great part of the NAD binding domain. Cross hybridization of chicken genomic DNA and mRNA with this human probe has been obtained in low-stringency conditions (Ittel et al., 1989). Using this 3’ probe and another one covering the 5’ region of the pADPRP human cDNA from nucleotide 1 to 697 (kindly provided by Dr G. Gradwohl), and called 5’ probe, we cloned and sequenced the chicken pADPRP cDNA. Its deduced aa sequence has been compared with the known mammalian as sequences.

Über schnell markierte nucleolare Desoxyribonucleinsäure in der perfundierten Rattenleber

In unseren vorhergehenden Versuchen1 haben wir festgestellt, das der Einbau von radioaktiven Vorlaufern, 3H-Thymidin und32PO 4 --- in die DNS der Nucleolarfraktion, welche nach einem leicht veranderten Verfahren2 von Allfrey und Mirsky 3 gewonnen wurde, viel hoher ist als im Rest der DNS des Zellkerns sowohl in der Rattenleber in vivo als in Hepatomzellen in vivo und in vitro. Seither wurde der rasche Einbau in Saugetierzellen-Nucleoli, unter anderem von der Arbeitsgruppe Granboulan 4, bestatigt, die das Problem vom elektronisch-mikroskopischen Standpunkt aus angefast hat. Man hat sich gefragt, was der hohe DNS-Turnover der Nucleolarfraktion zu bedeuten hat. Man nimmt allgemein an, das die Synthese der ribosomalen Ribonucleinsaure (RNS) im Nucleolus stattfindet. Spiegelmann u. Mitarb.5 haben einen Parallelismus zwischen der Zahl der Nucleoli und dem Prozentsatz der ribosomalen RNS, die sich mit nucleolarer DNS hybridieren last, gefunden.