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.

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.

CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus

1991 ◽  
Vol 11 (11) ◽  
pp. 5718-5726
Author(s):  
A Rowley ◽  
R A Singer ◽  
G C Johnston
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cycle Performance ◽  
Carboxyl Terminus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Negative Effects ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acid Protein ◽  
Mutant Cells

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals Evidence for two homologous, but nonidentical, Ia molecules determined by the I-EC subregion.

1979 ◽  
Vol 150 (1) ◽  
pp. 100-107 ◽  
Author(s):  
T L Delovitch ◽  
B H Barber
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Molecular Size ◽  
Gene Products ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Partial Digestion

Sequential immunoprecipitation, two-dimensional gel electrophoresis and peptide mapping analyses of B10A(3R), 35S-methionine-labeled, I-EC subregion products were performed. Evidence is presented here for the presence of two structurally homologous, but nonidentical, gene products of the I-EC subregion. These two Ia molecules are independently immunoprecipitable, identical in molecular size and charge, but differ by approximately equal to 20% in their peptides obtained by partial digestion with Staphylococcus aureus protease V8.

scholarly journals Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.

1996 ◽  
Vol 133 (1) ◽  
pp. 111-124 ◽  
Author(s):  
H A Sundberg ◽  
L Goetsch ◽  
B Byers ◽  
T N Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Immunofluorescent Staining ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nonpermissive Temperature ◽  
Carboxy Terminus ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Mutant Cells

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

scholarly journals The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function

1999 ◽  
Vol 19 (4) ◽  
pp. 2535-2546 ◽  
Author(s):  
Lynne D. Berry ◽  
Anna Feoktistova ◽  
Melanie D. Wright ◽  
Kathleen L. Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromosome Segregation ◽  
State Level ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Synthetic Lethal ◽  
C Complex ◽  
Mutant Cells

ABSTRACT The Schizosaccharomyces pombe dim1 + gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6mutant. At the restrictive temperature of 36°C, lid1-6mutant cells arrest with a “cut” phenotype similar to that ofcut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein ∼20S complex; the presence of lid1p in this complex depends upon functionalcut9 +. lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity oflid1 +. Further, lid1 +function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.

scholarly journals A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.

1988 ◽  
Vol 106 (4) ◽  
pp. 1171-1183 ◽  
Author(s):  
T Hirano ◽  
Y Hiraoka ◽  
M Yanagida
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Gradient Centrifugation ◽  
Metaphase Plate ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
In The Wild ◽  
Spindle Elongation ◽  
Mutant Cells

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

Cell differentiation in a temperature-sensitive stalkless mutant of Dictyostelium discoideum

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 235-243
Author(s):  
Aiko Amagai ◽  
Shuji Ishida ◽  
Ikuo Takeuchi
Keyword(s):  
Cell Differentiation ◽  
Dictyostelium Discoideum ◽  
Anterior Part ◽  
Cell Mass ◽  
Temperature Sensitive ◽  
Type Conversion ◽  
Spore Mass ◽  
Cell Type Conversion ◽  
Almost All ◽  
Mutant Cells

A temperature-sensitive aggregateless and stalkless mutant was isolated from Dictyostelium discoideum NC-4. The mutant cells cannot aggregate at 27°C, but aggregate and form normal fruiting bodies at 21°C. When the temperature was shifted to 27°C after aggregation at 21°C, almost all of the cells in the aggregate differentiated into spores. Neither stalk cells nor stalk tubes formed at 27°C. Inhibition of stalk formation was not lifted by addition of cyclic AMP. Nevertheless, the proportion of prespore to total cells within the mutant slugs was normal, at both 21 °C and 27 °C. At 27 °C, a slug was transformed into a spherical cell mass at the end of migration, within which pre-existing prespore cells differentiated into spores. The remaining prestalk cells were then converted to prespore cells which later became spores. As the cell-type conversion continued, formation of a spore mass resulted. The development of the mutant is thus consistent with the idea that the presumptive cell differentiation is directly related to the terminal cell differentiation. During migration at 27 °C, the number of prestalk cells decreased in the anterior part of the slug but instead increased at the foot or the rear part, whereas the prestalk—prespore pattern remained normal at 21 °C. The fact that a normal proportion of prespore cells was maintained in spite of their deranged distribution at 27 °C indicates that the regulation of proportion is independent of the formation of pattern.

Cell sorting out during the differentiation of mixtures of metabolically distinct populations of Dictyostelium discoideum

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 647-661
Author(s):  
C. K. Leach ◽  
J. M. Ashworth ◽  
D. R. Garrod
Keyword(s):  
Life Cycle ◽  
Dictyostelium Discoideum ◽  
Cell Sorting ◽  
Relative Number ◽  
Exponential Phase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Axenic Medium ◽  
Spore Population ◽  
The Relationship

The behaviour, during the multicellular phase of the life-cycle, of amoebae of Dictyostelium discoideum grown in different media is described. Amoebal populations were marked by growth-temperature-sensitive genetic lesions which do not interfere with developmental phenomena. The fate of cell populations was determined by measuring the relative number of mutant and wild-type cells at various stages in the life-cycle. Cells sort out during development in such a way that they may be ordered in a sequence in which those given early in the following list preferentially appear in the spore population when mixed with those given later in the list: cells grown in axenic medium + 86 mm glucose and harvested when in the exponential phase of growth; cells grown in axenic medium and harvested when in the exponential phase of growth; cells grown on bacteria and harvested when in the exponential phase of growth; cells grown in axenic medium + 86 mM glucose and harvested when in the stationary phase of growth. Chemotactic aggregation and grex migration are not essential for sorting-out to occur but, in the normal life-cycle, the cells of a grex formed from amoebae grown in different media have sorted out anteroposteriorly. The relationship between this sorting out behaviour and the mechanism of pattern formation in fruiting-body morphogenesis is discussed. Differences in density of the amoebae cannot account for the sorting out predispositions we observe.

Sign in / Sign up

Export Citation Format

Share Document