Purification and properties of the RuvA and RuvB proteins of Escherichia coli

Summary The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M_r ca. 100000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and -mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M_r ca. 85000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.     

A survey of lymphocyte chromosomal damage in Slovenian workers exposed to occupational clastogens.

Assays for sister-chromatid exchanges (SCE), unstable chromosome and chromatid aberrations and micronuclei were performed on blood lymphocytes from persons exposed protractedly to radiation or chemical hazards in the workplace. There was a general tendency with all endpoints examined for the yields to increase with years of working in the industry. This was especially marked for SCE. By comparison with a control group of administrative workers the levels of damage were higher, usually significantly so, in the occupational groups. These comprised workers at a nuclear research reactor, a hospital diagnostic X-ray department, a coal mine and a mercury ore mine.     

Lampyridae (Coleoptera): A plethora of mollicute associations

Beetles (Coleoptera) harbor many species ofAcholeplasma andSpiroplasma (division Tenericutes, class Mollicutes). Mollicutes were isolated from guts and/or hemocoels of firefly beetles (Lampyridae) from the United States (Maryland and West Virginia), Ecuador, and Tobago. Firefly beetles were frequent hosts for the group XIV spiroplasma, isolated from Ellychnia corrusca, and the group XIX spiroplasma, isolated fromPhoturis spp. The most unusual feature of the firefly-mollicute association is the carriage of four Mycoplasma species. Recent phylogenetic studies indicate that these species are members of a clade that includes a vertebrate pathogen,Mycoplasma mycoides. The high rate of occurrence ofMycoplasma species (which are, otherwise, infrequent in insects) in lampyrid beetles suggests that the association is significant. The unusual light-producing physiology of lampyrids (which is dependent on large pools of energy) and the production of large amounts of cardenolides from cholesterol (a critical growth factor for many mollicutes) may favor colonization by mollicutes. Offprint requests to: K. J. Hackett.     

Oligonucleotide site directed mutagenesis of all histidine residues within the T4 endonuclease V gene: role in enzyme-nontarget DNA binding

In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.     

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and ¹²⁵I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[¹²⁵I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of ¹²⁵I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of M_r 120 000 was internalized by macrophages somewhat more rapidly than copolymer of M_r 46 000, but was excluded from the yolk sac.     

ULTRASTRUCTURAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE IN THE OVULES OF SAINTPAULIA IONANTHA (GESNERIACEAE) AND ITS RELATION TO SYNERGID FUNCTION AND EMBRYO SAC NUTRITION

Ovules of African violet were analyzed for adenosine triphosphatase activity. Ovules from unpollinated flowers of three different ages were fixed in buffered, 3% paraformaldehyde, incubated in the Wachstein-Meisel medium, and processed for electron microscopy. Results showed a heavy reaction product in the endothelium and inner micropylar cells of the integument with decreasing amounts elsewhere. Reaction product was localized primarily on the plasma membrane, and occasionally in the nuclear membrane, endoplasmic reticulum, small vacuoles, and mitochondria. The synergids, egg, central cell, and antipodals were essentailly devoid of reaction product except for rare occurrences in the smaller vacuoles and mitochondria of the synergids, and fragmentary deposits on the plasma membrane of the antipodals. No differences were found in any of the floral stages examined. These results suggest that the integumentary cells nearest the embryo sac are equipped with the necessary enzymes for active translocation of solutes into the embryo sac and that the cells of the megagametophyte apparently function more passively in this regard.