* Physiologisch-chemisches Institut, Universität Tübingen, 72076 Tübingen, Germany; and Anatomisches Institut, Universität
Tübingen, 72072 Tübingen, Germany
A Saccharomyces cerevisiae mutant in cell division cycle gene CDC48 shows typical markers of apoptosis: membrane staining with annexin V, indicating an exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane; intense staining, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method, indicating DNA fragmentation; and chromatin condensation and fragmentation. The coordinate occurrence of these events at different locations in the cell, which have no obvious connection except their relation to apoptosis, implies the presence of the molecular machinery performing the basic steps of apoptosis already in yeast. Saccharomyces cerevisiae may prove a suitable model to trace the roots of apoptosis.
APOPTOSIS is a form of programmed cell death with an
important role in development and homeostasis of
metazoan organisms. Apoptosis allows the rapid
removal of unwanted or damaged cells that could otherwise inflame the surrounding cells with their cytoplasmic
contents. In contrast, during necrosis, a form of cell death
that results from overwhelming cellular injury, cells lyse
and release cytoplasmic material. The apoptotic program
is switched on in irreparably damaged or potentially dangerous cells such as self-reactive lymphocytes or cells that
have been infected by viruses. Furthermore, it is involved
in tumor suppression and in a wide range of diseases such
as AIDS, neurodegenerative processes, and ischemic stroke
(Steller, 1995 Apoptotic cells are characterized by a set of distinct
morphological changes (Kerr et al., 1972 The initiation of apoptosis is a highly coordinated and
regulated process. It can be induced or suppressed by a lot
of different intracellular and extracellular signals such as
Bcl-2 family proteins (Bax, Bak, and Bcl-2), IL-1 Though no proteins homologous to any of these apoptotic regulators have been detected in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, the
expression of Bax or p53 causes a severe effect on cell proliferation. Overexpression of human p53 inhibits cell growth
of S. pombe and S. cerevisiae as in higher eukaryotes, however it does not result in an apoptotic phenotype (Bischoff
et al., 1992 We investigated a mutant of baker's yeast in cell division cycle gene CDC48 for markers of apoptosis. Cdc48p
plays an important role in the homotypic fusion of the endoplasmic reticulum; in vitro, Cdc48p is the only soluble
protein necessary for the fusion of ER-derived vesicles
(Latterich et al., 1995 Cdc48p is a member of the AAA family, which is characterized by a highly conserved element of ~230 amino
acid residues, containing an ATP binding consensus sequence that can be present singly or in two copies. A database of the AAA family is available on the World Wide
Web at http://yeamob.pci.chemie.uni-tuebingen.de/.
We describe a cdc48 mutant of baker's yeast displaying
several of the characteristic morphological markers of apoptosis. This is the first indication that the basic machinery of
apoptosis is already present in unicellular lower eukaryotes.
Yeast Plasmids and Strains
YEp52/CDC48 was constructed by adding HindIII sites to CDC48 (directly before start ATG and 205 bp 3 YEp52/CDC48 or YEP52/cdc48S565G were transformed into strain
KFY247 (MAT a/MAT Cells were grown on YEP (1% yeast extract, 2% Bacto peptone) containing 4% glucose or 4% galactose, respectively. Cultures were inoculated to reach the stationary phase after 36-48 h of fermentation.
For nuclear staining, cells were washed with PBS, incubated with 1 µg/
ml diaminophenylindole (DAPI) in PBS for 10 min, and then rinsed three
times with PBS.
Electron Microscopy
Yeast cells were fixed with phosphate-buffered glutardialdehyde, cell
walls were removed, and the cells were postfixed with osmium tetroxide
and uranyl acetate, and then dehydrated as described by Byers and Goetsch
(1991) Terminal Deoxynucleotidyl Transferase-mediated
dUTP Nick End Labeling (TUNEL) Staining
DNA strand breaks were demonstrated by labeling free 3 Annexin V Staining
Exposed phosphatidylserine was detected by reaction with FITC-coupled
annexin V (ApoAlert Annexin V Apoptosis Kit; CLONETECH Laboratories, Inc., Palo Alto, CA). Yeast cells were washed in sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl2, 35 mM potassium phosphate, pH 6.8), digested
with 5.5% glusulase (Boehringer Mannheim) and 15 U/ml lyticase (Sigma
Chemical Co.) in sorbitol buffer for 2 h at 28°C, harvested, washed in
binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM
CaCl2; CLONETECH Laboratories, Inc.) containing 1.2 M sorbitol buffer, harvested and resuspended in binding buffer/sorbitol. 2 µl annexin-FITC (CLONETECH Laboratories, Inc.) and 2 µl propidium iodide (500 µg/ml) were added to 38 µl cell suspension, and then incubated for 20 min
at room temperature. The cells were harvested, suspended in binding
buffer/sorbitol, and applied to a microscopic slide.
A Yeast Mutant in Cell Division Cycle Gene CDC48
Accumulates Cells with Disrupted Chromatin
To evaluate the importance of various conserved residues
of Cdc48p, we changed a serine residue in the COOH-proximal AAA box of Cdc48p to a glycine residue (allele
cdc48S565G) by oligonucleotide-directed mutagenesis, and
put the mutated gene under the control of a GAL1 promoter. (The GAL1 promoter is induced by galactose in
the growth medium and repressed by glucose. CDC48
however, expressed from a glucose-repressed GAL1 promoter, enables cell proliferation, indicating a leaky repression.) The plasmid was transformed into a diploid yeast
(KFY247), heterozygously disrupted in CDC48, and transformants were sporulated. Spores containing the disrupted
cdc48::URA3 do not germinate on glucose medium and
need 7 d to form visible colonies on galactose medium. Control spores containing the same vector carrying a wild-type allele of CDC48 in addition to a disrupted chromosomal copy, form visible colonies within 2-3 d on both
glucose and galactose media. On glucose medium, cdc48S565G
segregants grow slowly (generation time 185 min instead
of 90 min for the control), and cease proliferating at 37°C
(ts phenotype). On galactose medium the segregants grow
like a CDC48 wild-type control.
Microscopic observation of stationary phase cultures of
these segregants shows abnormally elongated and misformed cells (Fig. 1) with the nuclear chromatin disrupted
into several fragments, reminiscent of apoptotic metazoan
cells. We systematically investigated the cdc48S565G strain,
KFY437, under different growth conditions for diagnostic markers of apoptosis.
Chromatin condensation and fragmentation is a typical
marker of apoptosis. Often, fragments are aligned as a ring
close to the nuclear envelope (Clifford et al., 1996
Electron microscopic investigation of exponentially
grown KFY437 cultures shows extensive chromatin condensation along the nuclear envelope (Fig. 3, b-d), as well
as cells containing nuclear fragments (Fig. 3, e and f). Endoplasmic reticulum is more prominent and its lumen appears wider than in wild-type controls (Fig. 3 a).
DNA Is Fragmented in cdc48 Mutant KFY437 after
Prolonged Growth
Apoptotic DNA cleavage produces free 3
Analysis of the isolated chromosomal DNA by agarose
electrophoresis did not show a DNA ladder (not shown),
that has been found in many apoptotic systems as the result of DNA cleavage between nucleosomes. This may be
caused by the S. cerevisiae chromatin structure with approximately no linker DNA between the nucleosomes (Lowary and Widom, 1989 cdc48 Mutant KFY437 Exposes Phosphatidylserine at
the Cytoplasmic Membrane
In mammalian cells, phosphatidylserine is predominantly
located on the inner leaflet of the plasma membrane, and
is translocated to the outer leaflet when apoptosis is induced (Martin et al., 1995 For detection of phosphatidylserine in the outer phase
of the cytoplasmic membrane, the cell wall was removed
by digestion with lyticase, and the spheroblasts were incubated with FITC-labeled annexin V. More than 70% of
the KFY437 cells grown exponentially on glucose medium
(Fig. 5 a) and ~50% of stationary phase cells grown on galactose medium show a strong fluorescence around the whole circumference of the cell. Control cultures of wild-type strains (Fig. 5 c), of cdc48 point mutants KFY415,
KFY416, and KFY438, of cdc48-3ts strain rE24-15 arrested
at 37°C, and of mutants in cell cycle genes cdc2, cdc19, and
cdc31 arrested at 37°C only show weak staining in the cell
lumen or no detectable fluorescence. 5-20% of the protoplasts, both from wild-type and mutant strains, take up
propidium iodide indicating membrane damage. These
cells all show a strong annexin V staining of the whole cell
and no peripheral ring (Fig. 5 b).
A yeast mutant in cell division cycle gene CDC48 shows a
number of morphological and molecular features that are
considered typical indicators of apoptosis in metazoan cells:
exposure of phosphatidylserine on the outer leaflet of the
cytoplasmic membrane, DNA breakage, chromatin condensation and fragmentation, and even the abnormal cell
morphology with a series of tiny buds, most of them lacking chromatin (Fig. 2 o/p, and q/r), can be considered the
equivalent of apoptotic bodies. The coordinate occurrence of these markers caused by a single point mutation implies
their connection even in yeast. None of the other alleles of
CDC48, and none of the cell division cycle-arrested mutants investigated shows any of these phenomena, indicating that they are not the inevitable consequence of any cell
division cycle arrest.
Apoptosis serves the purpose of removing unused or potentially harmful cells in higher organisms (Raff, 1992 It appears more likely that the connection between nuclear condensation, DNA fragmentation, the inversion of
the cytoplasmic membrane, and possibly the formation of
cell fragments is evolutionarily old, and was used for the
development of apoptosis by linking it to signaling pathways in metazoan organisms.
In mammals, exposition of phosphatidylserine serves as
bait for macrophages that then deal with the disposal of the
apoptotic bodies (Fadok et al., 1992 CDC48 participates in processes ensuring the integrity
of the endoplasmic reticulum and the nuclear envelope.
Aberrations in these structures caused by cdc48S565G might
trigger the processes observed. In wild-type cells of S. cerevisiae, endoplasmic reticulum is only scarcely seen in electron micrographs (Schwencke, 1991 The tools of yeast genetics and molecular biology are a
powerful means to identify the components of complex
physiological pathways. There is no clear picture of the
events following the action of the ICE proteases in apoptosis yet. Yeast might be a suitable model for clarification.
).
; Wyllie, 1980
;
Wyllie et al., 1980
). An early marker of apoptosis is the exposition of phosphatidylserine on the cell surface, whereas
it is normally concentrated in the luminal layer of the cytoplasmic membrane (Martin et al., 1995
). DNA is cleaved
between nucleosomes (Wyllie, 1980
) and the chromatin condenses (Kerr et al., 1972
), typically starting as a ring at the inner side of the nuclear envelope (Clifford et al.,
1996
). Finally, cells break up into membrane-enclosed fragments, the apoptotic bodies (Kerr et al., 1972
), which are
rapidly phagocytosed and digested by macrophages.
-converting enzyme (ICE)1 proteases (caspases), and p53. p53
is the most frequently altered gene in human cancers.
DNA damage induces p53 expression, which leads to cell
cycle arrest at G1 or to induction of apoptosis (Donehower
and Bradley, 1993
). Bax induces apoptosis in mammalian cells by the activation of ICE proteases (Chinnaiyan et al.,
1996
), which mediate the cleavage of several proteins including those of the nuclear matrix and nuclear envelope,
finally leading to DNA fragmentation.
). Expression of Bax in the yeast S. pombe is lethal, coexpression of the anti-apoptotic protein Bcl-2 suppresses this effect. As in mammals, Bcl-2 appears to inactivate Bax by forming mixed dimers: a mutant of Bcl-2 that
fails to heterodimerize with Bax does not rescue yeast cell
growth (Jürgensmeier et al., 1997
). However, Bax-induced
cell death in S. pombe is not accompanied by any classical
morphological feature of apoptosis. Neither evidence of
nuclear fragmentation nor of chromatin margination against the nuclear envelope, nor internucleosomal DNA fragmentation was observed. Jürgensmeier et al. (1997)
, therefore labeled the Bax-induced effect "cytotoxicity," to distinguish it from apoptosis.
). A defect in CDC48 results in a cell
cycle arrest as a large budded cell with the nucleus located
in the neck between the mother and the daughter cell at
16°C (Moir et al., 1982
; Fröhlich et al., 1991
).
Materials and Methods
of the stop codon) and cloning the
gene into the HindIII site of YEp52 (Broach et al., 1983
). The ChameleonTM site directed mutagenesis kit (Stratagene, Heidelberg, Germany) was used for site-directed mutagenesis of plasmid YEp52/CDC48. All mutations were confirmed by DNA sequencing. The codon of serine 565 of
CDC48 was mutagenized to a glycine codon (allele cdc48S565G, plasmid
YEP52/cdc48S565G) with oligonucleotide 5
-GGTATGGTGAAGGGGAATCTAACATCC-3
. For selection of mutant plasmids, a unique BglI site
in the bla gene of the vector was destroyed with oligonucleotide 5
-CCCTTCCAGCCGGCTGGTTTATTGC-3
.
CDC48/cdc48::URA3 his4-619/his4-619 leu3-3,112/leu2-3,112 ura3-52/ura3-52) and the transformants were sporulated;
segregants containing the cdc48::URA3 disruption were recognized by
their ability to grow without external uracil. Segregant KFY437 (MAT a
cdc48::URA3 his4-619 leu3-3,112 ura3-52 YEp52/cdc48S565G) was used for
all cytologic experiments. Segregant KFY417 (MAT a cdc48::URA3 his4-619 leu3-3,112 ura3-52 YEp52/CDC48) with a wild-type CDC48 on YEp52,
and isogenic strain KFY439 with intact CDC48 (MAT a CDC48 his4-619
leu3-3,112 ura3-52) were used as wild-type controls. Cell division cycle
mutants used as controls were Hartwell (1973) strains LH12021 (cdc31ts),
arresting with an elongated nucleus in a budded cell; LH369 (cdc1ts), LH395
(cdc19ts), LH321 (cdc25ts), and LH17048 (cdc29ts), all arresting unbudded;
LH370 (cdc2ts), LH244 (cdc9ts), and LH386 (cdc20ts), and strains DBY2028
(MAT a ade2 lys2 leu2 ura3 cdc46ts), and rE24-15 (MAT
his4-619 cdc48-3ts; Moir et al., 1982
), all arresting with a large bud. Strains with a disrupted chromosomal copy of CDC48 and with cdc48 alleles, cdc48S565T
(strain KFY438), cdc48Y834E (strain KFY415), or cdc48Y834F (strain KFY416),
expressed from vector YEp52 were used as controls. Strains KFY438 and
KFY415 grow like wild type, KFY416 shows a doubled generation time on
glucose-containing media, resembling cdc48S565G strain KFY437 in this respect.
for stationary phase cells. After the 100% ethanol washes, cells
were washed with 100% acetone, infiltrated with 50% acetone/50% Epon
for 30 min and with 100% Epon for 20 h. Cells were transferred to fresh
100% Epon and incubated at 56°C for 48 h before cutting thin sections
and staining with lead acetate.
-OH termini
with FITC-labeled deoxyuridine, which was detected with alkaline phosphatase-coupled, anti-fluorescein antibody, and the formation of a dye
precipitate with a phosphatase substrate (In Situ Cell Death Detection Kit,
AP; Boehringer Mannheim, Mannheim, Germany). Yeast cells were fixed
with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and
Pringle, 1984
). The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min on ice,
rinsed twice with PBS, incubated with 10 µl TUNEL reaction mixture
(200 U/ml terminal deoxynucleotidyl transferase, 10 mM FITC-labeled
dUTP, 25 mM Tris/HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim) for 60 min at 37°C, rinsed three times with PBS, incubated with 50 µl Converter AP solution (alkaline phosphatase- labeled, anti-FITC antibody; Boehringer Mannheim) for 30 min at 37°C,
rinsed three times with PBS, and stained by incubation with 50 µl naphthol) AS-MX phosphate (Sigma Chemical Co., Munich, Germany), 0.8 mg/ml, fast red TR salt (Sigma Chemical Co.), 1 mg/ml, 2% dimethylformamide, 1 mM levamisole in 100 mM Tris/HCl, pH 8.2, for 30 min at room
temperature. A coverslip was mounted with a drop of Kaiser's glycerol
gelatin (Merck, Darmstadt, Germany).
Results
Fig. 1.
Morphology of yeast mutant strain KFY437. Stationary
phase cells of KFY437 (a-d) and of control strain KFY417 (e)
grown on YEP/glucose for 5 d after inoculation (phase contrast).
Bars: (a) 10 µm; (b-e) 10 µm.
[View Larger Version of this Image (54K GIF file)]
). DAPI-stained KFY437 cells from exponentially growing as well as
from stationary cultures on glucose medium show a continuous DNA ring within the nucleus in 1-10% of the cells,
DNA fragments arranged as a ring at the inner side of the
nuclear envelope in 1-20% of the cells, and several randomly distributed nuclear fragments in 10-50% of the cells
(Fig. 2). Fragmentation increases with incubation time and after 7 d on glucose medium, 80% of the cells have an abnormal chromatin distribution. In KFY437 cells grown
exponentially on galactose, <10% of the cells show chromatin fragmentation. In stationary cultures from galactose
medium, 40% of the cells have a fragmented nucleus.
Stationary wild-type strains show the nucleus as a single
round spot in all cells, their morphology does not change
markedly during prolonged incubation (Fig. 2 a). cdc48 point mutants, KFY415, KFY416, and KFY438, show unfragmented nuclei both during exponential growth and in
the stationary phase. cdc48-3ts strain rE24-15 arrested at
37°C shows an unfragmented nucleus in the neck between
mother and daughter cells (Fig. 2 b). Cell division cycle
mutants in genes cdc1, cdc19, cdc20, cdc25, cdc29, and
cdc31 that were arrested at 37°C show unfragmented nuclei. Mitochondria appear as dots of far less brightness and
size than the nuclei or most nuclear fragments, and are
predominantly located near the border of the cells.
Fig. 2.
Chromatin fragmentation. DAPI stain (c-n, p, and r)
and phase contrast representation (o, and q) of KFY437 cells,
and DAPI stain of KFY417 (a) and of cdc48 mutant rE24-15 (b)
as controls. i-k were cells grown exponentially on YEP/glucose;
a, c-h, and l-n are stationary cells from glucose medium, harvested 2 d after inoculation; o/p and q/r are stationary cells from
glucose medium, harvested 5 d after inoculation; b shows exponentially growing cells of rE24-15 that were cell division cycle arrested by a 3-h incubation at 37°C. Bar, 10 µm.
[View Larger Version of this Image (121K GIF file)]
Fig. 3.
Electron micrographs of yeast mutant strain
KFY437. Wild-type control
KFY417 (a) and mutant
KFY437 (b-f) grown exponentially on YEP/glucose for
24 h. N, nucleus; V, vacuole;
and M, mitochondria. Endoplasmic reticulum is marked
by arrowheads; chromatin
condensation is marked by arrows. Bars, 1 µm.
[View Larger Version of this Image (142K GIF file)]
-OH termini,
which can be detected by labeling with modified nucleotides (e.g., DIG-dUTP) catalyzed by terminal deoxynucleotidyl transferase. The TUNEL (Gavrieli et al., 1992
; Gorczyca et al., 1993
) method is a fast and sensitive way to
visualize the amount of DNA fragmentation in individual
cells. At the end of the exponential growth phase, both
on galactose and on glucose containing media, >80% of
KFY437 cells have an intensive red nuclear stain with the
TUNEL assay, corresponding to a strong fragmentation
(Fig. 4 a). Early exponential cultures of KFY437 contain
~20% TUNEL-positive cells. Control cultures of wild-type
cells (Fig. 4 b), of cdc48 point mutants KFY415, KFY416,
and KFY438, of cdc48-3ts strain rE24-15 arrested at 37°C,
and of mutants in cell cycle genes cdc2, cdc9, cdc19, cdc31,
and cdc46 arrested at 37°C, show unstained or only slightly
red nuclei. An aliquot of the protoblasts used for TUNEL
staining was tested for integrity by incubation with 23 µg/
ml propidium iodide. Less than 3% of the protoplasts became stained, proving that the DNA fragmentation is not
a result of cell necrosis.
Fig. 4.
DNA strand breakage visualized by TUNEL staining.
Cells of KFY437 (a) and of control strain KFY417 (b) grown on
YEP/glucose to the end of the exponential growth phase (36 h after inoculation) were stained for DNA strand breaks with
TUNEL. Bar, 10 µm.
[View Larger Version of this Image (141K GIF file)]
). In addition, a number of cell
types undergo apoptosis without the occurrence of a DNA
ladder, even when chromatin condensation occurs (Oberhammer et al., 1993
).
). Phosphatidylserine exposure
serves as a sensitive marker for early stages of apoptosis. It
can be detected with annexin V, which binds to phosphatidylserine with high affinity in the presence of Ca2+. As in
mammalian cells, S. cerevisiae has an asymmetric distribution of phospholipids within the cytoplasmic membrane,
90% of the phosphatidylserine is oriented towards the cytoplasm (Cerbón and Calderón, 1991
).
Fig. 5.
Exposition of phosphatidylserine at the cytoplasmic
membrane. Cells of KFY437 (a and b) and of control strain
KFY417 (c) grown exponentially for 12 h on YEP/glucose were
stained with FITC-labeled annexin V for detection of exposed
phosphatidylserine (a and c) and propidium iodide for detection
of damaged cells (b). Bar, 10 µm.
[View Larger Version of this Image (31K GIF file)]
Discussion
). Even
in a unicellular organism like yeast, such a mechanism
would be of evolutionary advantage. If a cell sensing impending fatal damage stopped proliferating and consuming resources, it would improve the chances for its clonal
relatives. However, the fact that similar observations with
yeast have not been published before suggests that this is a
rare event. In addition, a database search of the complete
S. cerevisiae genome shows no potential homologue for
genes involved in the triggering of apoptosis in metazoans:
yeast contains neither members of the ICE protease family (caspases), nor p53, nor genes with similarity to ced-4,
bcl-2, or bax.
; Martin et al., 1995
).
Our observation in yeast indicates a more fundamental
connection between phosphatidylserine exposure and other
events of apoptosis. Therefore, the exposure probably has
not been developed to attract macrophages, but rather phagocytic cells throughout evolution have learned to interpret the phenomenon as a signal of attraction.
). An electron microscopic observation of the mutant shows that endoplasmic
reticulum accumulates and that its lumen appears dilated
as compared to wild-type endoplasmic reticulum. This could
be a direct effect of the mutation, ultimately leading to the
apoptotic phenotype. The phenotype is allele specific; none of the other cdc48 mutant alleles investigated cause
any of the apoptotic effects.
Received for publication 25 April 1997 and in revised form 1 August 1997.
Address all correspondence to Kai-Uwe Fröhlich, Physiologisch-Chemisches Institut, Hoppe-Seyler-Strasse 4, Universität Tübingen, 72076 Tübingen, Germany. Tel.: 49-7071-297-3360. Fax: 49-7071-296-6390. E-mail: kaifr{at}uni-tuebingen.deWe thank D. Mecke for discussion and support, H. Schwarz for generous advice, and S. Sigrist for help in preparing the figures. We are grateful to J. Gatfield for critical reading of the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft.
ICE, IL-1-converting enzyme;
TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling.
1. | Adams, A.E.M., and J.R. Pringle. 1984. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J. Cell Biol. 98: 934-945 [Abstract]. |
2. | Bischoff, J.R., D. Casso, and D. Beach. 1992. Human p53 inhibits growth in Schizosaccharomyces pombe. Mol. Cell. Biol. 12: 1405-1411 [Abstract]. |
3. | Broach, J.R., Y.-Y. Li, L.-C.C. Wu, and M. Jayaram. 1983. Vectors for high-level, inducible expression of cloned genes in yeast. In Experimental Manipulation of Gene Expression. M. Inouye, editor. Academic Press, London. 83-117. |
4. | Byers, B., and L. Goetsch. 1991. Preparation of yeast cells for thin-section electron microscopy. Methods. Enzymol. 194: 602-608 |
5. | Cerbón, J., and V. Calderón. 1991. Changes of the compositional asymmetry of phospholipids associated to the increment in the membrane surface potential. Biochim. Biophys. Acta. 1067: 139-144 |
6. | Chinnaiyan, A.M., K. Orth, K. O'Rourke, H. Duan, G.G. Poirier, and V.M. Dixit. 1996. Molecular ordering of the cell death pathway. J. Biol. Chem. 171: 4573-4576 . |
7. |
Clifford, J.,
H. Chiba,
D. Sobieszczuk,
D. Metzger, and
P. Chambon.
1996.
RXR![]() |
8. | Donehower, L.A., and A. Bradley. 1993. The tumor suppressor p53. Biochim. Biophys. Acta. 1155: 181-205 |
9. |
Fadok, V.A.,
D.R. Voelker,
P.A. Campbell,
J.J. Cohen,
D.L. Bratton, and
P.M. Henson.
1992.
Exposure of phosphatidylserine on the surface of apoptotic
lymphocytes triggers specific recognition and removal by macrophages.
J.
Immunol.
148:
2207-2216
|
10. | Fröhlich, K.-U., H.-W. Fries, M. Rüdiger, R. Erdmann, D. Botstein, and D. Mecke. 1991. Yeast cell cycle protein CDC48p shows full length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression. J. Cell Biol. 114: 443-453 [Abstract]. |
11. | Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119: 493-501 [Abstract]. |
12. | Gorczyca, W., J. Gong, and Z. Darzynkiewicz. 1993. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53: 1945-1951 [Abstract]. |
13. |
Hartwell, L.H.,
R.K. Mortimer,
J. Culotti, and
M. Culotti.
1973.
Genetic control
of the cell-division cycle in yeast: V. Genetic analysis of cdc mutants.
Genetics.
74:
267-286
|
14. | Jürgensmeier, J.M., S. Krajewski, R.C. Armstrong, G.M. Wilson, T. Oltersdorf, L.C. Fritz, J.C. Reed, and S. Ottilie. 1997. Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe. Mol. Biol. Cell. 8: 325-339 [Abstract]. |
15. | Kerr, J.F., A.H. Wyllie, and A.R. Currie. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26: 239-257 |
16. | Latterich, M., K.-U. Fröhlich, and R. Schekman. 1995. Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell. 82: 885-893 |
17. | Lowary, P.T., and J. Widom. 1989. Higher-order structure of Saccharomyces cerevisiae chromatin. Proc. Natl. Acad. Sci. USA. 86: 8266-8270 [Abstract]. |
18. | Martin, S.J., C.P.M. Reutelingsperger, A.J. McGahon, J.A. Rader, R.C.A.A. van Schie, D.M. LaFace, and D.R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182: 1545-1556 [Abstract]. |
19. |
Moir, D.,
S.E. Steward,
B.C. Osmond, and
D. Botstein.
1982.
Cold-sensitive
cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies.
Genetics.
100:
547-563
|
20. | Oberhammer, F., J.W. Wilson, C. Dive, I.D. Morris, J.A. Hickman, A.E. Wakeling, P.R. Walker, and M. Sikorska. 1993. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO (Eur. Mol. Biol. Organ.) J. 12: 3679-3684 [Abstract]. |
21. | Raff, M.C.. 1992. Social controls on cell survival and cell death. Nature (Lond.). 356: 397-400 |
22. | Schwencke, J. 1991. Vacuoles, internal membraneous systems and vesicles. In The Yeasts. Vol. 4. A.H. Rose, and J.S. Harrison, editors. Academic Press, London. 347-432. |
23. | Steller, H.. 1995. Mechanisms and genes of cellular suicide. Science (Wash. DC). 267: 1445-1449 |
24. | Wyllie, A.H.. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (Lond.). 284: 555-556 |
25. | Wyllie, A.H., J.F.R. Kerr, and A.R. Currie. 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68: 251-306 |