Altered poly(ADP-ribose) metabolism in family members of patients with systemic lupus erythematosus.

OBJECTIVE: To determine whether the previously detected decrease in poly(ADP-ribose) synthesis in systemic lupus erythematosus (SLE) is familial. METHODS: Poly(ADP-ribose) metabolism was studied in the peripheral blood lymphocytes of members of 3 families with lupus and in healthy controls. RESULTS: Synthesis, as determined by the incorporation of 3H NAD into acid precipitable counts was 30% of normal in the patients with SLE (p < 0.001) and 70% of normal in the unaffected family members (p < 0.05). Family members also had increased prevalence of antinuclear antibodies but the presence of these antibodies did not correlate with abnormalities in poly(ADP-ribose) metabolism. CONCLUSION: The healthy biologic relatives of patients with SLE have a decrease in poly(ADP-ribose) synthesis similar to, but less marked than, their affected relatives. Poly(ADP-ribose) polymerase activity appears to be an inherited trait and abnormalities in it may be one of the susceptibility factors for SLE. If this is the case, then further investigation of this gene and its regulation is warranted.

Decreased mRNA levels coding for poly(ADP-ribose) polymerase in lymphocytes of patients with SLE.

Poly(ADP-ribose) metabolism is altered in patients with SLE. In order to localize the defect, the levels of poly(ADP-ribose) polymerase-specific mRNA were measured from dot blots of total RNA from peripheral blood lymphocytes. In this preliminary study, eleven patients with SLE and two with antiphospholipid syndrome were compared to three controls. It was found that the mean levels of specific mRNA were ten fold lower in the PBL from SLE patients compared to controls and no overlap of values was seen between the two groups. No such decrease was seen in the PBL from the patients with antiphospholipid syndrome. It is concluded that the defect in poly(ADP-ribose) polymerase metabolism that is seen in SLE patients occurs at the level of transcription or mRNA turnover.

Triplex DNA in plasmids and chromosomes.

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.

Altered metabolism of poly(ADP-ribose) in the peripheral blood lymphocytes of patients with systemic lupus erythematosus.

A method was developed to measure poly(ADP-ribose) metabolism in peripheral blood lymphocytes. The technique involved the isolation of lymphocytes on Ficoll gradients, followed by lysis with 5M NaCl. The synthesis and degradation of poly(ADP-ribose) in this crude lysate, measured by the incorporation of 3H-labeled NAD into acid-precipitable counts, was compared in 18 patients with systemic lupus erythematosus (SLE), 10 patients with rheumatoid arthritis, and in 10 control patients without rheumatoid arthritis. Patients with SLE showed a 70% decrease in poly(ADP-ribose) synthesis (P less than 0.001); this decreased synthesis persisted even with the addition of histones or DNase. We present possible explanations of the role of poly(ADP-ribose) in SLE.

The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.

Increased synthesis of poly(ADP-ribose) in isolated liver nuclei from autoimmune NZB/NZW mice.

The synthesis and degradation of poly(ADP-ribose) were investigated in isolated liver nuclei from autoimmune NZB/W mice and four strains of normal mice. Compared to normal mice the maximum levels of incorporation of [3H]NAD into poly(ADP-ribose) were increased about 2-fold in the autoimmune mice. The kinetics of incorporation suggested that this change was due to an increase in the activity of the polymerase rather than a decrease in the level of degradative enzymes. Thus there may be a connection between autoimmunity and poly(ADP-ribose) metabolism.

A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.