scholarly journals Selection of chemotaxis mutants of Dictyostelium discoideum.

1987 ◽  
Vol 104 (1) ◽  
pp. 151-161 ◽  
Author(s):  
J E Segall ◽  
P R Fisher ◽  
G Gerisch
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Population ◽  
Selection Procedure ◽  
Total Cell ◽  
Wild Type ◽  
Total Cell Population ◽  
Mutant Cells ◽  
Efficient Selection ◽  
Filter Layer ◽  
Selection Of

A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant-degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described.

Pattern formation in Dictyostelium discoideum

Development ◽  
1977 ◽  
Vol 40 (1) ◽  
pp. 215-228
Author(s):  
D. Forman ◽  
D. R. Garrod
Keyword(s):  
Life Cycle ◽  
Pattern Formation ◽  
Dictyostelium Discoideum ◽  
Cell Population ◽  
Growth Conditions ◽  
Total Cell ◽  
Immunofluorescent Staining ◽  
Fruiting Bodies ◽  
Fluorescent Intensity ◽  
Total Cell Population

Immunofluorescent staining of the prespore cells of the cellular slime mould Dictyostelium discoideum was carried out using a heterologous spore antibody. The highly specific staining of the prespore vesicles (PSVs) within the prespore cells enabled quantitative determinations to be made of the rate and extent of development of these cells throughout the life cycle. The results showed that PSVs first appeared in a large proportion of the cells shortly after the cells had chemotactically aggregated into multicellular masses. During the later phases of the life cycle, the proportion of cells containing PSV increased, as did the fluorescent intensity of their PSVs, until the early culmination stage of development when 85–90 % of the total cell population contained PSVs. Lowering the temperature of development delayed the onset of vesicle formation and decreased the proportion of prespore cells in the total cell population. Changing the growth conditions of the cells prior to multicellular development also had a significant effect on the proportions of prespore cells, as did the use of a mutant known to give rise to fruiting bodies with a reduced number of spores. The comparability between these estimates of prespore cell proportions at culmination and previously reported spore:stalk ratios within fruiting bodies confirms the view that PSVs are reliable indicators of prespore cells. The finding that temperature and growth conditions and the use of mutants all of which are known to affect spore:stalk ratios, also all affected prespore proportions in the expected direction, adds further weight to this argument. The fact that prespore cells are beginning to differentiate early in the multicellular phase of the life cycle and the related finding that such differentiation always precedes formation of the grex tip are results of considerable importance to the development of a model for pattern formation in D. discoideum.

scholarly journals Total Cell Counts of the Bone Marrow of Normal Albino Rats from 1 to 50 Weeks of Age

Blood ◽  
1959 ◽  
Vol 14 (4) ◽  
pp. 409-414 ◽  
Author(s):  
WILLIAM T. BURKE ◽  
CHARLES HARRIS
Keyword(s):  
Bone Marrow ◽  
Cell Population ◽  
Normal Values ◽  
Age Groups ◽  
Considerable Change ◽  
Total Cell ◽  
Albino Rats ◽  
Cell Counts ◽  
Total Cell Population ◽  
Femoral Bone Marrow

Abstract A method is described by which the total nucleated cell count of femoral bone marrow of the rat can be estimated and cell population expressed in terms of differential counts. Normal values of total nucleated cell counts and the cellular distributions are given for seven age groups. These data indicate considerable change in bone marrow total cell population in rats one to 10 weeks of age.

scholarly journals Isoprenylcysteine Carboxy Methylation Is Essential for Development in Dictyostelium discoideum

2007 ◽  
Vol 18 (10) ◽  
pp. 4106-4118 ◽  
Author(s):  
Ying Chen ◽  
Kyle J. McQuade ◽  
Xiao-Juan Guan ◽  
Peter A. Thomason ◽  
Michael S. Wert ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Protein Subunit ◽  
Encoding Gene ◽  
Carboxy Terminal ◽  
Mutant Cells ◽  
Do So

Members of the Ras superfamily of small GTPases and the heterotrimeric G protein γ subunit are methylated on their carboxy-terminal cysteine residues by isoprenylcysteine methyltransferase. In Dictyostelium discoideum, small GTPase methylation occurs seconds after stimulation of starving cells by cAMP and returns quickly to basal levels, suggesting an important role in cAMP-dependent signaling. Deleting the isoprenylcysteine methyltransferase-encoding gene causes dramatic defects. Starving mutant cells do not propagate cAMP waves in a sustained manner, and they do not aggregate. Motility is rescued when cells are pulsed with exogenous cAMP, or coplated with wild-type cells, but the rescued cells exhibit altered polarity. cAMP-pulsed methyltransferase-deficient cells that have aggregated fail to differentiate, but mutant cells plated in a wild-type background are able to do so. Localization of and signaling by RasG is altered in the mutant. Localization of the heterotrimeric Gγ protein subunit was normal, but signaling was altered in mutant cells. These data indicate that isoprenylcysteine methylation is required for intercellular signaling and development in Dictyostelium.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals Fenhexamid - an efficient and inexpensive fungicide for selection of Magnaporthe oryzae transformants

2021 ◽  
Author(s):  
Alex Wegner ◽  
Louisa Wirtz ◽  
Thomas Leisen ◽  
Matthias Hahn ◽  
Ulrich Schaffrath
Keyword(s):  
Magnaporthe Oryzae ◽  
Model Organism ◽  
Selection Procedure ◽  
Phytopathogenic Fungi ◽  
Infection Process ◽  
Ergosterol Biosynthesis ◽  
Fusarium Fujikuroi ◽  
Plant Pathogen Interactions ◽  
Efficient Selection ◽  
Selection Of

AbstractMagnaporthe oryzae is one of the most economically important phytopathogenic fungi, and is used as a model organism to study plant-pathogen interactions. To unravel the infection process, forward and reverse genetic approaches are essential, but are often hindered by the lack of a straightforward selection procedure for transformants. Here we report on the use of fenhexamid, an inhibitor of ergosterol biosynthesis, for selection of M. oryzae transformants. An allele of the sterol 3-ketoreductase gene of Fusarium fujikuroi (FfERG27), known to confer resistance to fenhexamid, has already been used successfully with transformants of Botrytis cinerea. Our results demonstrate that expression of the FfERG27 allele in M. oryzae also enables highly efficient selection of transformants on fenhexamid-containing media. The use of fenhexamid is an inexpensive alternative for selection as compared to commonly used antibiotics like hygromycin. No impact on growth and infection phenotypes of fenhexamid resistant M. oryzae mutants was detected, which underpins its usefulness for selecting M. oryzae transformants.

scholarly journals Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum.

1991 ◽  
Vol 11 (6) ◽  
pp. 3171-3179 ◽  
Author(s):  
S Alexander ◽  
S Leone ◽  
E Ostermeyer
Keyword(s):  
Dictyostelium Discoideum ◽  
Translational Control ◽  
Suppressive Effect ◽  
Developmental Expression ◽  
Wild Type ◽  
Rna Analysis ◽  
Type Pattern ◽  
Bona Fide ◽  
Lectin Protein ◽  
Mutant Cells

Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription.

Erythropoiesis in peripheral blood of tuatara (Sphenodon punctatus) and turtle (Malaclemys terrapin)

1970 ◽  
Vol 48 (2) ◽  
pp. 209-212 ◽  
Author(s):  
Vibeke E. Engelbert ◽  
Ann Dorothy Young
Keyword(s):  
Cell Population ◽  
Peripheral Blood ◽  
Total Cell ◽  
Red Cells ◽  
Malaclemys Terrapin ◽  
Domestic Birds ◽  
Total Cell Population ◽  
Sphenodon Punctatus ◽  
Clone Cells

Erythropoiesis in peripheral blood of domestic birds was shown earlier to arise directly from the nucleus of mature erythrocytes as nuclear protuberations that later broke free. Three to seven percent of new red cells originated in this way from clone cells.Investigations of peripheral blood in reptiles has further demonstrated formation of nucleated red cells as clones from nuclear buds of mature erythrocytes. Clone cells plus new immature red cells constitute, in Sphenodon punctatus, 8–18% and, in turtle, Malaclemys terrapin, 18–25% of the total cell population. Some lymphocytes and granulocytes appear also to arise from nuclear buds. Thrombocytes were present in some animals, absent in others, but were not counted in the present work.

SPHEROPLAST INDUCTION AND LYSIS OF BCG STRAINS BY GLYCINE AND LYSOZYME

1966 ◽  
Vol 12 (2) ◽  
pp. 255-261 ◽  
Author(s):  
H. Sato ◽  
B. B. Diena ◽  
L. Greenberg
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Population ◽  
Lithium Chloride ◽  
Synthetic Medium ◽  
Total Cell ◽  
Cell Populations ◽  
Standard Curve ◽  
Total Cell Population

Spheroplast induction and lysis of 6 BCG strains of Mycobacterium tuberculosis by glycine and lysozyme was studied in various media. Spheroplast production was noted in only three strains involving 20% of cell populations. Lysis, as distinct from spheroplast induction, occurred in Dubos medium containing 1.5% glycine and 0.01% lysozyme after 24–48 hours of incubation. Estimation from a standard curve indicated 40 to 70% lysis of the total cell population after 7–10 days of incubation.Similarly, lysis of BCG cells occurred when the inducers, glycine, lysozyme, and lithium chloride, were added to nitrogen-starved cultures grown in Aldridge synthetic medium for 7 to 8 days.

scholarly journals TOTAL CELL NUMBER IN FETAL BRAIN

2011 ◽  
Vol 19 (1) ◽  
pp. 35 ◽  
Author(s):  
Grethe Badsberg Samuelsen ◽  
Nenad Bogdanović ◽  
Henning Laursen ◽  
Niels Graem ◽  
Jørgen Falck Larsen ◽  
...  
Keyword(s):  
Birth Weight ◽  
Cell Population ◽  
Fetal Brain ◽  
Fold Increase ◽  
Cell Number ◽  
Total Cell ◽  
Cortical Plate ◽  
Total Cell Number ◽  
Normal Birth ◽  
Total Cell Population

In this study the material comprises brains from three aborted fetuses and two fullterm infants who died at birth.The gestational ages ranged from the 22nd week to term. All cases were without malformations, known chromosomal abnormality, hydrops, and systemic infections, and all had normal birth weights with fetal growth indices (observed birth weight/expected mean birth weight) between 0.9 - 1.05. The preliminary results show a five fold increase in the total cell population in the marginal zone/cortical plate, MZ/CP (future neocortex), from week 22 until term. In the transient subplate zone, SP, the total cell number was more than doubled from week 22 to week 30-35, and then decreased towards term. In the intermediate zone, IZ (future white matter), the total cell population was doubled from week 22 until term. The total cell number in the entricular/subventricular zone, VZ/SZ (germinal matrix), was reduced by a factor of five from week 22 until term. A histological differentiation between neurons and glial cells was not possible. The optical fractionator was used to estimate the total cell population in four characteristic developmental zones in the human fetal brain. Fetal brain tissue undergoes considerable and rather unpredictable shrinkage during fixation. However, using the fractionator principle it is possible to eliminate this problem, provided that the structure of interest (one brain hemisphere) is fully intact.

Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum

1991 ◽  
Vol 11 (6) ◽  
pp. 3171-3179
Author(s):  
S Alexander ◽  
S Leone ◽  
E Ostermeyer
Keyword(s):  
Dictyostelium Discoideum ◽  
Translational Control ◽  
Suppressive Effect ◽  
Developmental Expression ◽  
Wild Type ◽  
Rna Analysis ◽  
Type Pattern ◽  
Bona Fide ◽  
Lectin Protein ◽  
Mutant Cells

Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription.

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