Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.     

Protein dynamics from chemical shift and dipolar rotational spin-echo 15N NMR

The partial collapse of dipolar and chemical shift tensors for peptide NH and for the amide NH at cross-link sites in cell wall peptidoglycan, of intact lyophilized cells of Aerococcus viridans, indicates NH vector root-mean-square fluctuations of 23 degrees. This result is consistent with the local mobility calculated in typical picosecond regime computer simulations of protein dynamics in the solid state. The experimental root-mean-square angular fluctuations for both types of NH vectors increase to 37 degrees for viable wet cells at 10 degrees C. The similarity in mobilities for both general protein and cell wall peptidoglycan suggests that one additional motion in wet cells involves cooperative fluctuations of segments of cell walls, attached proteins, and associated cytoplasmic proteins.     

Ca2+ ionophores inhibit superoxide generation by chemotactic peptide in rabbit neutrophils and the correlation with intracellular calcium

Summary Effects of Ca²⁺ ionophores, A23187 and lasalocid, on superoxide anion generation by chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, in rabbit peritoneal exudate neutrophils were studied. The ionophores by themselves did not activate superoxide anion generation in these neutrophils. When preincubated with the cells for 2 min, both the ionophores inhibited superoxide generation induced by chemotactic peptide. The inhibition was present even in the absence of extracellular Ca²⁺ and the inhibition was better then. Lasalocid produces a dose-dependent chlortetracycline fluorescence decrease response in neutrophils loaded with chlortetracycline. This response is independent of extracellular Ca²⁺ concentration and is related to release of Ca²⁺ from intracellular storage sites. The dose-range at which lasalocid gives this response is same as the dose-range at which it causes inhibition of superoxide response. It may be concluded that the inhibition of superoxide generation by these ionophores is correlated to intracellular Ca²⁺ modulation.Abbreviations FMLP Formyl-Methionyl-Leucyl-Phenylalanine methyl ester     

Rhythmic signalling and entrainment inVespa orientalis larvae: Characterization of the underlying interactions

Rhythmic “circa-second” contrations of larvae of the hornetVespa orientalis, believed to serve as hunger signals, were studied. A considerable degree of coordination among individual larvae, both in frequency and phase of these contractions, has been observed. The oscillations of singly isolated larvae are of short duration, non-constant, with increasing intervals in between and there is a substantial variability in the patterns shown by different larvae. In contrast, the association of two or more larvae leads to enhancement of their periodic behaviour and to (partial) entrainment. Communication among larvae may perhaps be mediated by the sound pulses (“scratching” noises) which are generated by these contractions. We have subjected individual and grouped larvae to external sound pulses and were able to demonstrate: (a) enhancement of rhythmic activity; (b) phase resetting; (c) entrainment to an external oscillator within a range of frequencies; (d) the existence of a subharmonic mode of entrainment. We propose a simple phenomenologic model to account for these larvae responses. Our model assumes the existence of an “energy” variable which declines with time but is upgraded, in a phase-dependent way, by external stimuli. Based in part on work performed by V. Barenholz-Paniry in partial fulfillment of the requirements for the M.Sc. degree from the Sackler Faculty of Medicine, Tel Aviv University, 1986.     

Association of poly(A) polymerase with U1 RNA

Previous studies (Stetler, D. A., and Jacob, S. T. (1984) J. Biol. Chem. 259, 7239-7244) have shown that poly(A) polymerase from adult rat liver (liver-type) is structurally and immunologically distinct from the corresponding rat hepatoma (tumor-type) enzyme. When hepatoma 7777 (McA-RH 7777) cells were labeled with [32P]inorganic phosphate, followed by immunoprecipitation with anti-hepatoma poly(A) polymerase antibodies and analysis of the RNAs in the immunoprecipitate, only one labeled small nuclear RNA corresponding to U1 RNA was found. Preimmune sera did not form a complex with U1 RNA. Hepatoma poly(A) polymerase antisera did not immunoprecipitate U1 RNA or any other small nuclear RNA from a cell line (H4-11-EC3) which does not contain the tumor-type poly(A) polymerase. Immunoblot analysis of hepatoma 7777 nuclear extract or purified poly(A) polymerase with anti-ribonucleoprotein antisera did not show any cross-reactivity of the latter sera with poly(A) polymerase. The major RNA immunoprecipitated from the hepatoma nuclear extracts using trimethyl cap (m3G) antisera corresponded to the RNA immunoprecipitated with poly(A) polymerase antisera. These data indicate that U1 RNA is closely associated with poly(A) polymerase and suggest the potential involvement of this RNA in the cleavage/polyadenylation of mRNA precursor.     

The Tangled Bank: The maintenance of sexual reproduction through competitive interactions

The maintenance of sexual reproduction is discussed using a model based on the familiar Lotka-Volterra competition equations. Both the equilibrium and the stability conditions that allow a sexual population to resist invasion by a single asexual clone are considered. The equilibrium conditions give results similar to previous models: When the cost of sex, within phenotype niche width, and environmental variance are low, the sexual population coexists with the asexual clone and remains at a high density. However, the asexual clone is never completely excluded. Analysis of the stability conditions shows a different picture: The introduction of an asexual clone considerably reduces the stability of the community. However, owing to its larger total niche width, the sexual population exists partly in a “competitor-free space” where the asexual clone has almost no influence on the outcome of the interactions. Therefore the asexual clone is less stable than the sexual population and has a higher probability of extinction. In contrast, the sexual population does not become extinct, since the extreme phenotypes remain at a stable, though low, density, and the central phenotypes, where stability is low, are recreated every generation through recombination. I therefore conclude that the ecological conditions under which sexual reproduction is favored over asexual reproduction are fairly easily attained and are more general than previous analyses had suggested.     

Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2K^k and H-2D^d MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2–5)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.