Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Crystallization of the complex of human IFN-γ and the extracellular domain of the IFN-γ receptor

A complex of human interferon-γ (IFN- γ) with the soluble extracellular domain of the IFN- γ receptor α-chain (IFN-γ-R) has been crystallised. Crystals of the complex were grown using PEG 4000 as the precipitating agent in the presence of β-octyl glucoside. The receptor-ligand complex crystallizes in a monoclinic space group and diffracts to about 3.0 Å resolution. Isomorphous crystals have been obtained with complex containing selenomethionine and cysteine mutants of IFN-γ, which may facilitate the ongoing X-ray structure determination. © 1995 Wiley-Liss, Inc.     

The sensitivity of halobacteria to antibiotics

Abstract Eleven species of the genera Halobacterium, Halococcus and the recently proposed Haloarcula were tested by the macro-broth dilution method for their sensitivity to 20 antibiotics with different modes of action. The most active were bacitracin, erythromycin, haloquinone, rifampicin and novobiocin. Resistant mutants of H. mediterranei to bacitracin, chloramphenicol and josamycin were obtained with frequencies of spontaneous mutation between 10⁻⁴ and 10⁻⁷.     

Regulation of nitrogen fixation (nif) genes of Azospirillum brasilense by nifA and ntr (gln) type gene products

Abstract Eight Nif⁻ mutants of Azospirillum brasilense were obtained by N -nitrosoguanidine mutagenesis and isolated by growth on glutamate medium. Three of these mutants had no nitrogenase activity, possessed no nitrogenase structural proteins and were complemented by Klebsiella pneumoniae nifA . Evidence will be presented that one of these mutants is defective in a nifA type regulatory gene but the other two were also complemented by K. pneumoniae ntrC and may be ntrC⁻ -type mutants. A fourth mutant was defective in the MoFe component protein of nitrogenase.     

Phenotypic changes in hydrogen evolving chemostat cultures of Rhodopseudomonas capsulata

Abstract Spontaneous nitrogenase-negative (Nif⁻ mutants of Rhodopseudomonas capsulata were observed to accumulate with time in ammonium- or glutamate-limited chemostat cultures.
Nif⁻ mutants were characterized by their inability to grow under N₂ and to reduce acetylene or produce hydrogen gas when grown on glutamate. They lacked the nitrogenase structural proteins as evidenced by immunological techniques. On the other hand, no significant differences were found in the pathways of ammonia assimilation between the Nif⁻ mutants and the wild-type strain. The Nif⁻ mutants seem to result from a mutation in a regulatory gene.     

Mechanism of suppression in Drosophila. V. Localization of the purple mutant of Drosophila melanogaster in the pteridine biosynthetic pathway

The suppressible eye color mutant purple (pr) of Drosophila melanogaster is known to be unable to synthesize a wild-type complement of pteridine eye pigments. This study measures the reduced levels of drosopterins, sepiapterin, and an unidentified presumed pteridine in pr and pr ^bw. Pteridine analyses in double mutants combining pr with one of three other eye color mutants sepia, Henna-recessive³, and prune², suggest that the metabolic block in pr occurs prior to sepiapterin biosynthesis. Measurements of GTP and GTP cyclohydrolase in pr showed wild-type levels and indicate the metabolic block in pr to be at one of the steps converting dihydroneopterin triphosphate to sepiapterin. Quantitation of pteridines in suppressed purple [su(s) ²; pr and pr; su(pr) ^e3] shows restoration of pteridines to wild-type or nearly wild-type levels.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. VI. Enzymatic studies of two mutants unable to convert inosinic acid to adenylic acid

Ade^–H and ade^–I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade^–H did not have any detectable adenylosuccinate synthetase activity and ade^–I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.This work was supported by research grants from the National Institute of Aging (AG00029) and the National Foundation, March of Dimes (1-423), and by a contract from the Center for Toxicological Research, Food and Drug Administration (72-213). David Patterson is a recipient of a Research Career Development Award from the National Institute of Arthritis, Metabolic and Digestive Diseases (AM00044).Contribution (No. 218) from the Eleanor Roosevelt Institute for Cancer Research.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae. III. Nitrous acid.

Nitrous acid (NA) induced mutations efficiently in mitDNA, conferring resistance to erythromycin and weakly induces mit- mutations. In some strains of yeast it also enhanced rho- mutations. The frequencies of nuclear and mitochondrial mutations induced with NA are compared.     

The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus

The initiation of sporangiophores of Phycomyces was analyzed under oxygen-limiting conditions. Mutants lacking -carotene have a higher oxygen threshold than the wild type depending on the residual amount of -carotene. The supersensitivity to low oxygen tension is specific for sporangiophore initiation and can be suppressed by addition of either retinal, retinol or retinol acetate to the medium. It is suggested that retinol is a natural regulator of differentiation in Phycomyces.