Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.     

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.     

Proceedings of the fifty-fourth Annual Meeting of the American Association of physical anthropologists to be held in Knoxville,Tennessee—Holiday Inn World's fair April 10–13, 1985

Empirical analyses and models of the lineal effects of fission indicate that considerable genetic differentiation may occur at the time of group formation, thus confusing the usual positive relationship between historical affiliation and genetic differentiation. We analyze the effects of fission pattern on variation in highly heritable morphological traits among eight social groups on Cayo Santiago. The analysis is performed using general network autocorrelation methods that quantitatively and directly measure the amount of variation in social group mean morphology that can be explained by fission. All of the fission autocorrelation coefficients are strongly negative, indicating that groups most recently formed by fission are most dissimilar. Also, most of the variation between groups can be explained by the fission pattern, indicating that lineal fission is the most important process generating between-group variation on Cayo Santiago.     

Mice coisogenically immunized against H-2 class I antigens on transfected l cells reject transplanted embryonal carcinoma cells

Evidence is presented of the ability of H-2 class I antigens to function as teratocarcinoma transplantation (Gt) antigens. Coisogenic immunization against H-2 class I antigens expressed on transfected L cells is shown to induce resistance to embryonic carcinoma (EC) cell allografts. The K^b, D^b, D^d, and, in appropriate recipients, L^d antigens can function as Gt antigens. The protocol presented may be useful for the molecular identification of other genes encoding histocompatibility antigens.     

Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor

We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg²⁺ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg²⁺. We also tested for the effects of Mg²⁺ on detergent extractablity of pigment-protein complexes from intact membranes. Mg²⁺ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.Abbreviations LHC light harvesting complex - OG octyl glucoside - RC reaction center This paper is dedicated to Professor G. Drews on the occasion of his 60th birthday     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

Immunofluorescent localization of a murine seminal vesicle proteinase inhibitor

The indirect immunofluorescent technique was used to localize a proteinase inhibitor isolated from murine seminal vesicles. The inhibitor was found in the lumen and in the apical epithelium of the seminal vesicle but not in the testes, epididymides, ductus deferens or Cowper's glands. It was also associated with the anterior acrosomal region of ejaculated sperm and sperm recovered from the female tract within 5 min of coitus. The inhibitor is removed from uterine sperm between 2 to 4 h postcoitus, however sperm recovered from the uterus 2 h postcoitus will rebind inhibitor. The inhibitor is not normally associated with epididymal or ductus sperm although these sperm will bind purified inhibitor in vitro.     

Protein Patterns of Growing and Starved Cells of a Marine Vibrio sp.

Fingerprint protein patterns were produced by two-dimensional polyacrylamide electrophoresis on lysed cells of a Vibrio sp., Ant-300, which were prepared from growing and starved cultures. The cells were labeled with [³⁵S]methionine during growth and subsequently starved for up to 30 days. Samples were taken at selected time points representing stages in the starvation-survival process. Unlabeled starved cells were allowed to recover in the presence of [³⁵S]methionine to determine protein changes associated with the recovery from starvation. All growth conditions produced similar protein fingerprints; however, some protein spots disappeared, whereas others were seen only during starvation.