Nuclear plasmids in the Dictyostelium slime molds

Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2-microns circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3-27 kb and in copy number from 50-300 molecules per cell. Plasmids have been identified in approximately one-fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10(-4) per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.     

Mutagen-induced fetal anomalies and death following treatment of females within hours after mating

In an earlier study (Generoso et al., 1987), it was observed that the mutagen, ethylene oxide (EtO), produced remarkable increases in the incidence of developmental abnormalities and death of fetuses when early zygotic stages were exposed. This is a major finding in experimental induction of embryopathy, implicating genetic damage to the zygotes as the likely cause. In the subsequent study reported here, 3 other mutagens--ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), and triethylene melamine (TEM), were studied for embryopathic effects following exposure of dictyate oocytes, prefertilization oviducal eggs and sperm, early pronuclear zygotes, zygotes undergoing pronuclear DNA synthesis, and two-cell embryos. All 4 mutagens produced developmental abnormalities among living fetuses following exposure of early pronuclear zygotes (the only stage studied for this endpoint in this report). With respect to stage specificity and gestational timing of death of conceptuses, EMS and EtO on one hand and ENU and TEM on the other, are very similar to one another. EMS, like EtO, produced a high incidence of midgestation and late fetal deaths only in prefertilization oviducal eggs and sperm and in early pronuclear eggs. In contrast, ENU and TEM produced high losses of conceptuses in all postmating stages studied but death occurred primarily prior to or around the time of implantation. Thus, the frequency of induction and the expression of embryopathy, which ranged from early embryonic preimplantation and late fetal deaths to subtle fetal anomalies, are dependent upon the stage exposed and the mutagen used.     

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.     

Post-embedding methods for immunolocalization of elastin and related components in tissues

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.     

Protection of mice against mixed fission neutron-gamma (n: gamma = 1:1) irradiation by WR-2721, 16,16-dimethyl PGE2, and the combination of both agents

The survival of mice after whole-body exposure to a modified fission neutron-gamma field (n: gamma = 1:1) was used to examine radiation protection by WR-2721, 16,16-dimethyl PGE2(DiPGE2), and the combination of both agents. Administration of WR-2721 (453 mg/kg) increased the LD50/30 from 5.24 to 7.17 Gy (DMF = 1.37), whereas pretreatment with DiPGE2 (1.6 mg/kg) increased the LD50/30 to 5.77 Gy (dose modification factor (DMF) = 1.10). The combination of 453 mg/kg WR-2721 and 0.4 mg/kg DiPGE2 resulted in an LD50/30 of 7.33 Gy, yielding a DMF of 1.39. However, no significant difference in protection was obtained with the combination of the two agents compared to that seen with WR-2721 alone.