Studies of the diphtheria toxin receptor on chinese hamster cells

Concanavalin A, wheat germ agglutinin and the ovalbumin glycopeptide are all inhibitors of the cytotoxic effect of diphtheria toxin on Chinese hamster cells. Ovalbumin glycopeptide loses its inhibitory property after treatment with β-N-acetylglucosaminidase. This demonstrates the importance of the glycopeptide structure for the mechanism of inhibition. The glycopeptide may be a toxin cell-surface receptor analogue. Diphtheria toxin-resistant mutants were isolated in order to search for cells that might have an altered toxin receptor. One mutant was 10-to 15-fold more resistant to diphtheria toxin than wild-type cells when protein synthesis was measured as a function of toxin concentration. However, when protein synthesis was measured as a function of time at a high toxin concentration, the time before onset of inhibition was identical in the mutant and wild-type cells. We present evidence indicating that the resistance of this mutant can be accounted for by a decreased affinity of toxin for a cell-surface receptor.     

Comparison of in vivo and in vitro ribosomal RNA synthesis in nucleolar mutants ofXenopus laevis

Summary Cells of embryos carrying a lethal nucleolar mutation have been maintained in vitro for extended periods of time. Normally these mutants live only 9 to 12 days after fertilization but their cells in culture will survive for more than 3 months. The extent of ribosomal RNA (rRNA) synthesis was determined in primary cultures prepared from normal embryos and nucleolar mutants having different numbers of ribosomal RNA genes. We found that the accumulation of radioactivity into rRNA for normal and mutant embryos was similar in vivo and in vitro. In primary cultures of normal embryos which have two nucleoli per cell and mutant embryos which have only one nucleolus per cell, the incorporation of radio-activity into rRNA was similar even though the normal cells have twice as many rRNA genes. Thus the mechanism which regulates dosage compensation of the rRNA genes operates both in vivo and in vitro. This work was supported by Grant GB38651 from the National Science Foundation.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.     

Genetic alleviation of the self-fertilization complication when hybridizing monoecious Gelidium vagum

Fundamental genetic studies were initiated for the monoecious red alga Gelidium vagum. Color and sterility mutants were isolated and characterized to provide genetic tools, initially to identify hybrid plants when they occurred in crosses, and secondarily to eliminate self-fertilization altogether. When fertility phenotypes were scored, rapid onset of reproduction in culture was favored by long day-length, moderately high irradiance levels from fluorescent lights, warm temperature and the addition of Tris buffer to the medium. A recessive green mutant (designated grn 1) was characterized and used in subsequent crosses to allow a clear distinction between non-hybrid (green) and hybrid (red) offspring. Additional color mutants and a variety of reproductive mutants were also isolated and characterized. Male-sterile mutants had phenotypes ranging from apparently normal plants to those that produced no spermatia. Female-sterile mutants also included a variety of phenotypes, some plants having post-fertilization malfunctions during the development of the carposporophyte. Only a fraction of the sterility mutations have been phenotypically or genetically characterized, but some are straightforwardly inherited as stable, nuclear, single-gene defects. From the genetic recombination pattern, one female-sterile mutant may be loosely linked (39 cMorgans) to the grn 1 marker gene. Male sterility very effectively eliminated selfing without affecting the production of carpospores in crosses, thereby overcoming one of the most serious genetic difficulties in working with this monoecious species.     

Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisme

Summary Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RP021, also called RPBI). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RP021 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to –8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five is alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the is mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RP021 and RP026, the gene encoding the 17.9 kDa subunit shared by RNA polymerases 1, 11, and III was investigated by determining the ability of increased dosage of RP026 to suppress the is phenotype imposed by rpo21-4 to –8. Suppression of the is defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RP026 subunit of RNA polymerase II.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Involvement of photorespiration and glycolate pathway in carbonic anhydrase induction and inorganic carbon concentration in Chlamydomonas reinhardtii

Interaction between induction of carbonic anhydrase (CA) activity, induction of inorganic carbon (C_i) concentrating mechanisms and the photorespiratory glycolate pathway has been studied in wild type 6145c and photorespiratory mutant 18–7F (low in phosphoglycolate phosphatase activity) cells of C. reinhardtii . Cell transfer from high CO₂ (5%, v/v) to low CO₂ (0.03%) provoked an increase of extracellular and total (extracellular plus intracellular) CA in both wild type and mutant cells. During adaptation to low CO₂ conditions, both strains excreted ammonium to the medium at a similar rate in the presence of l -methionine- d-l -sulfoximine (MSX), an inhibitor of glutamine synthetase (GS). MSX also provoked ammonium excretion by air adapted wild type and mutant cells, even though both strains had high levels of CA activity and of C_i concentrating activities.
GS increased in both strains after transfer from high to low CO₂ conditions. However, this increase was abolished by aminooxyacetate, an inhibitor of the glyoxylate-serine aminotransferase, and by glycolaldehyde, an inhibitor of triose phosphate to ribulose 1,5-bisphosphate conversion. CA synthesis did not occur in the presence of either aminooxyacetate or glycolaldehyde. Algae grown in high CO₂ in the presence of aminooxyacetate did not induce C_i concentrating mechanisms. Integration of these three processes, i.e., CA synthesis, C_i-concentration, and photorespiratory glycolate pathway is proposed in the framework of carbon metabolism of the alga.     

Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody

Dense-core granules represent an adaptation of specialized secretory cell to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tet-rahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants. © 1992 Wiley-Liss, Inc.