scholarly journals Motility mutants of Dictyostelium discoideum.

1982 ◽  
Vol 93 (3) ◽  
pp. 705-711 ◽  
Author(s):  
S C Kayman ◽  
M Reichel ◽  
M Clarke
Keyword(s):  
Oxygen Consumption ◽  
Dictyostelium Discoideum ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Track Formation ◽  
Genetic Events ◽  
Axenic Growth

We describe six motility mutants of Dictyostelium discoideum in this report. They were identified among a group of temperature-sensitive growth (Tsg) mutants that had been previously isolated using an enrichment for phagocytosis-defective cells. The Tsg mutants were screened for their ability to produce tracks on gold-coated cover slips, and several strains were found that were temperature-sensitive for migration in this assay. Analysis of spontaneous Tsg+ revertants of 10 migration-defective strains identified six strains that co-reverted the Tsg and track formation phenotypes. Characterization of these six strains indicated that they were defective at restrictive temperature in track formation, phagocytosis of bacteria, and pseudopodial and filopodial activity, while retaining normal rates of oxygen consumption and viability. Because they had lost this group of motile capabilities, these strains were designated motility mutants. The Tsg+ revertants of these mutants, which coordinately recovered all of the motile activities, were found at frequencies consistent with single genetic events. Analysis of the motility mutants and their revertants suggests a relationship between the motility mutations in some of these strains and genes affecting axenic growth.

motA1552, a mutation of Dictyostelium discoideum having pleiotropic effects on motility and discoidin I regulation.

Genetics ◽  
1988 ◽  
Vol 118 (3) ◽  
pp. 425-436
Author(s):  
S C Kayman ◽  
R Birchman ◽  
M Clarke
Keyword(s):  
Linkage Group ◽  
Dictyostelium Discoideum ◽  
Growth Temperature ◽  
Segregation Analysis ◽  
Pleiotropic Effects ◽  
Temperature Sensitive ◽  
Temperature Induction ◽  
Group Ii ◽  
Unstable Mutations ◽  
Axenic Growth

Abstract The Dictyostelium discoideum mutant MC2 exhibits temperature-sensitive growth, temperature-sensitive motility, and temperature induction of discoidin I synthesis. These three phenotypes of MC2 were not separated in the genetic experiments reported here. They were therefore assigned to the mutation motA1552, which was mapped to linkage group II by segregation analysis and by analysis of mitotic recombinant diploids. In one motA1552 strain, loss of motility preceded accumulation of discoidin I by 3 hr, indicating that discoidin I is not involved in generation of the motility defect. Expression of motA1552 phenotypes varied both among strains carrying the mutation, and among different clones of a particular strain. MC2 and its derivatives displayed elevated levels of recombination between whiA and acrA on linkage group II, and yielded highly unstable mutations at the acrA locus. Accumulation of large amounts of discoidin I during axenic growth of strain AX3 was found to depend on the presence of a second linkage group II mutation, daxA1551. This mutation was already present in the strain mutagenized to isolate motA1552, complicating explication of motA1552 action.

scholarly journals Overproduction of discoidin I by a temperature-sensitive motility mutant of Dictyostelium discoideum.

1984 ◽  
Vol 4 (6) ◽  
pp. 1035-1041 ◽  
Author(s):  
S Biswas ◽  
S C Kayman ◽  
M Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Axenic Medium ◽  
Two Dimensional Gel Electrophoresis ◽  
Motility Mutant ◽  
And Migration ◽  
Mutant Cells

Dictyostelium discoideum MC2 is a temperature-sensitive motility mutant of AX3. Mutant cells are incapable of growth, phagocytosis, and migration under restrictive conditions (Kayman et al., J. Cell Biol. 92:705-711, 1982). We show here that at the restrictive temperature MC2 cells grown axenically or on bacteria synthesized excessive quantities of the lectin discoidin I. By two-dimensional gel electrophoresis and peptide mapping, the proteins overproduced by MC2 cells were indistinguishable from discoidin I synthesized at lower levels in AX3 cells. At least two of the three species of discoidin I were overproduced. This protein family constituted 9% of the total protein in cells that were incubated overnight at 27 degrees C in axenic medium. Although MC2 cells are defective in nutrient uptake under restrictive conditions, the overproduction of discoidin I did not appear to be part of a pleiotropic response to starvation. We propose that transcription of the coordinately regulated discoidin I genes is altered in mutant cells. This alteration may be related to the motility defects manifested by MC2.

scholarly journals Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

1993 ◽  
Vol 4 (9) ◽  
pp. 931-939 ◽  
Author(s):  
D Feldheim ◽  
K Yoshimura ◽  
A Admon ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Functional Characterization ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Restrictive Temperature ◽  
Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

scholarly journals Saccharomyces cerevisiae HOC1, a Suppressor of pkc1, Encodes a Putative Glycosyltransferase

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 637-645 ◽  
Author(s):  
Aaron M Neiman ◽  
Vijay Mhaiskar ◽  
Vladimir Manus ◽  
Francis Galibert ◽  
Neta Dean
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Lysis ◽  
Integral Membrane Protein ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Calcofluor White ◽  
Kinase C

The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

Sulfate suicide selection of Dictyostelium discoideum mutants defective in protein glycosylation

1986 ◽  
Vol 6 (8) ◽  
pp. 2820-2827
Author(s):  
J A Boose ◽  
E J Henderson
Keyword(s):  
Dictyostelium Discoideum ◽  
Developmental Regulation ◽  
Selection Procedure ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Metabolic Labeling ◽  
Replica Plating ◽  
Oligosaccharide Processing ◽  
Glycoprotein Processing

The assembly and processing of glycoprotein-linked oligosaccharides in Dictyostelium discoideum has been shown to generate a wide array of glycan structures which undergo dramatic developmental regulation. As late steps in processing of these oligosaccharides involve sulfation, a sulfate suicide selection procedure was developed to select for temperature-sensitive glycoprotein-processing mutants. Of 673 clones derived from the survivors of suicide selection, 99 were classified by replica-plating fluorography as temperature sensitive for sulfate transport or incorporation. Of these, 74 were unable to complete the developmental program to the fruiting body stage at the restrictive temperature, 29 being blocked in some aspect of aggregation and 45 being blocked at some postaggregation stage. Quantitative metabolic labeling experiments with representative clones showed that they incorporated wild-type levels of [35S]methionine but reduced levels of sulfate at the restrictive temperature. The specific incorporation patterns in the mutants suggest that distinct oligosaccharide-processing steps are involved in different developmental events.

scholarly journals Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype.

1985 ◽  
Vol 5 (5) ◽  
pp. 907-915 ◽  
Author(s):  
N S Atkinson ◽  
R W Dunst ◽  
A K Hopper
Keyword(s):  
Rna Processing ◽  
Elevated Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Recessive Lethal ◽  
Recessive Lethal Mutation ◽  
Sensitive Allele

The RNA1 gene product is believed to be involved in RNA metabolism due to the phenotype of a single conditionally lethal, temperature-sensitive allele, rna1-1. We cloned the RNA1 gene and determined that it produces a 1,400-nucleotide polyadenylated transcript. On a multicopy plasmid, the mutant rna1-1 allele partially complements the rna1-1 temperature-sensitive growth defect. This suggests that the temperature-sensitive nature of the rna1-1 allele results from the synthesis of a product with lowered activity or stability at elevated temperatures or from a decrease in synthesis of the rna1-1 product at the restrictive temperature. A chromosomal disruption of RNA1 behaves as a recessive lethal mutation. Haploids bearing the disruption were isolated by sporulating a diploid heterozygous for the disrupted allele and the rna1-1 allele and possessing an episomal copy of the RNA1 gene. Analysis of the rescued haploids bearing the chromosomal disruption indicated that the recessive lethal phenotype of the RNA1 disruption is not merely due to a block in spore germination. Unexpectedly, diploids heterozygous for the disruption and the rna1-1 alleles become aneuploid for chromosome XIII at a frequency of 2 to 5%. It appears that the disrupted RNA1 allele on a multicopy plasmid also promotes aneuploidy for chromosome XIII. Promotion of aneuploidy seems to be a phenotype of this particular allele of RNA1.

scholarly journals Sulfate suicide selection of Dictyostelium discoideum mutants defective in protein glycosylation.

1986 ◽  
Vol 6 (8) ◽  
pp. 2820-2827 ◽  
Author(s):  
J A Boose ◽  
E J Henderson
Keyword(s):  
Dictyostelium Discoideum ◽  
Developmental Regulation ◽  
Selection Procedure ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Metabolic Labeling ◽  
Replica Plating ◽  
Oligosaccharide Processing ◽  
Glycoprotein Processing

The assembly and processing of glycoprotein-linked oligosaccharides in Dictyostelium discoideum has been shown to generate a wide array of glycan structures which undergo dramatic developmental regulation. As late steps in processing of these oligosaccharides involve sulfation, a sulfate suicide selection procedure was developed to select for temperature-sensitive glycoprotein-processing mutants. Of 673 clones derived from the survivors of suicide selection, 99 were classified by replica-plating fluorography as temperature sensitive for sulfate transport or incorporation. Of these, 74 were unable to complete the developmental program to the fruiting body stage at the restrictive temperature, 29 being blocked in some aspect of aggregation and 45 being blocked at some postaggregation stage. Quantitative metabolic labeling experiments with representative clones showed that they incorporated wild-type levels of [35S]methionine but reduced levels of sulfate at the restrictive temperature. The specific incorporation patterns in the mutants suggest that distinct oligosaccharide-processing steps are involved in different developmental events.

scholarly journals CHARACTERIZATION OF DOMINANT RESISTANCE TO COBALT CHLORIDE IN DICTYOSTELIUM DISCOIDEUM AND ITS USE IN PARASEXUAL GENETIC ANALYSIS

Genetics ◽  
1978 ◽  
Vol 90 (1) ◽  
pp. 37-47
Author(s):  
Keith L Williams
Keyword(s):  
Dictyostelium Discoideum ◽  
Cobalt Chloride ◽  
Resistance Mutation ◽  
Differential Sensitivity ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Cobaltous Chloride ◽  
Chloride Resistance ◽  
Resistant Strains

ABSTRACT Strains of Dictyostelium discoideum resistant to cobaltous chloride have been isolated at a frequency of approximately 10-6. The resistant strains have one of three phenotypes, recessive to wild type, dominant to wild type and dominant to wild type but requiring the presence of cobaltous chloride to maintain resistance. Strains carrying a dominant cobaltous chloride resistance mutation and a recessive growth temperature-sensitive mutation can be mixed with wild-type haploid lines and then subjected to selection so that only diploid lines survive. Differential sensitivity to cycloheximide has also been observed. Hypersensitivity to cycloheximide in combination with dominant cobaltous chloride resistance provides a means of selecting diploids without the use of temperature-sensitive mutations.

Overproduction of discoidin I by a temperature-sensitive motility mutant of Dictyostelium discoideum

1984 ◽  
Vol 4 (6) ◽  
pp. 1035-1041
Author(s):  
S Biswas ◽  
S C Kayman ◽  
M Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Axenic Medium ◽  
Two Dimensional Gel Electrophoresis ◽  
Motility Mutant ◽  
And Migration ◽  
Mutant Cells

Dictyostelium discoideum MC2 is a temperature-sensitive motility mutant of AX3. Mutant cells are incapable of growth, phagocytosis, and migration under restrictive conditions (Kayman et al., J. Cell Biol. 92:705-711, 1982). We show here that at the restrictive temperature MC2 cells grown axenically or on bacteria synthesized excessive quantities of the lectin discoidin I. By two-dimensional gel electrophoresis and peptide mapping, the proteins overproduced by MC2 cells were indistinguishable from discoidin I synthesized at lower levels in AX3 cells. At least two of the three species of discoidin I were overproduced. This protein family constituted 9% of the total protein in cells that were incubated overnight at 27 degrees C in axenic medium. Although MC2 cells are defective in nutrient uptake under restrictive conditions, the overproduction of discoidin I did not appear to be part of a pleiotropic response to starvation. We propose that transcription of the coordinately regulated discoidin I genes is altered in mutant cells. This alteration may be related to the motility defects manifested by MC2.

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