Copyright1998 by the Genetics Society of America
Regulation of Septum Formation in Aspergillus nidulans
by a DNA Damage Checkpoint Pathway
Steven D. Harris and Peter R. Kraus
Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030-3205 Manuscript received September 6, 1997
Accepted for publication November 17, 1997
ABSTRACT
In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimXactivity to control the timing of septum formation.
T
HE fidelity of cellular reproduction is dependent negative regulatory sites on the 20-kD myosin light chainupon the temporal coordination of cytokinesis with protein by p34cdc2may delay cytokinesis until the
initia-nuclear division. Recent evidence suggests that continu- tion of anaphase (Satterwhiteet al. 1992). However,
ous signaling between midzone microtubules and the the importance of this mechanism for the temporal
cellular cortex is required for the completion of cyto- regulation of cytokinesis in animal cells is still open to
kinesis in animal cells (Wheatley and Wang 1996; question (FishkindandWang1995). In S. cerevisiae, the
Oegema andMitchison1997). A number of proteins reorganization of the actin cytoskeleton at the incipient
that localize to the spindle midzone during telophase division site is triggered by the destruction of the
Clb-could conceivably mediate these signaling events. This Cdc28 cdk complex (LewandReed1993). The
destruc-includes chromosomal passenger proteins such as the tion of p34cdc2 also appears to promote cytokinesis in
inner centromere proteins (INCENPs; Cooke et al. fission yeast, since a mutation that causes premature
1987; Earnshaw and Mackay 1994), telophase disc loss of cdk activity leads to multiple rounds of septation
proteins such as TD-60 (Andreassenet al. 1991;Mar- (Fankhauseret al. 1993). Collectively, these studies
sug-tineau et al. 1995), kinesin-related motor proteins gest that cdk activity suppresses cytokinesis until the
(Williamset al. 1995), and anillin (FieldandAlberts
initiation of anaphase.
1995). Interactions between mitotic nuclei and the cel- The multicellular filamentous fungus Aspergillus
nidu-lular cortex also appear to be required for the comple- lans undergoes cytokinesis by forming crosswalls termed
tion of cytokinesis in the unicellular fungi Schizosaccharo- septa. The process of septum formation is tightly coordi-myces pombe and Saccharocoordi-myces cerevisiae (Fankhauseret
nated with nuclear division in A. nidulans hyphal cells.
al. 1993;MuroneandSimanis1996;Yanget al. 1997).
For example, as in animal cells, mitotic nuclei are likely
Notably, inappropriate activation of the spindle-assem- to specify the division site (Wolkowet al. 1996).
Further-bly checkpoint appears to directly inhibit septum
forma-more, recent observations indicate that continuous
sig-tion in S. pombe (MuroneandSimanis1996).
naling between adjacent mitotic spindles and the cortex In both animal and fungal cells, cyclin-dependent
is required for formation of the contractile actin ring kinase (cdk) activity has been implicated in controlling
and the completion of septation (MomanyandHamer
the timing of cytokinesis relative to nuclear division.
1997). Previous results have shown that uninucleate co-In Xenopus lysates, phosphorylation of three different
nidia give rise to multinucleate hyphal cells
(predivi-sional hyphal cells;Harris 1997) by delaying septum
formation until they satisfy a size control and complete
Corresponding author: Steven Harris, Department of Microbiology,
a subsequent mitotic division (Wolkowet al. 1996). The
University of Connecticut Health Center, 263 Farmington Avenue,
Farmington, CT 06030-3205. E-mail: sharris@nso2.uchc.edu uncoupling of cytokinesis from nuclear division in these
All genetic manipulations were performed as described
pre-cells has permitted the identification of a class of
tem-viously (Harriset al. 1994). sepB nimXcdc2AF, sepJ nimXcdc2AF, sepA
perature-sensitive (TS) mutants with a distinctive
phe-nimXcdc2AF, and sepB uvs double mutants were identified as TS
notype suggestive of a defect in the coordination of segregants, which were also sensitive to 5 mm HU.
septation with nuclear division (Morris 1976;Harris Growth conditions:For all experiments, YGV or CM1
sup-plements containing conidia at a density of 1–53104/ml was
et al. 1994). Although these mutants (sepB3, sepB4, and
gently poured into petri dishes containing glass coverslips.
sepJ1) grow to a sufficient extent and are capable of
The conidia settled to the bottom of the petri dish and adhered
undergoing multiple rounds of nuclear division, they
tightly to the coverslips. At the appropriate times, coverslips
fail to form septa. In addition, sepB mutants also exhibit with adherent cells were removed and processed for
micros-defects in chromosomal DNA metabolism, which lead copy.
Wild-type strains were incubated at either 288 or 378. HU
to enhanced levels of chromosome nondisjunction and
(used at concentrations ranging from 1–10 mm) and
diepoxy-mitotic recombination (Harris and Hamer 1995).
octane (DEO; Aldrich Chemical Co., Milwaukee, used at
con-These phenotypes suggested that sepB mutants fail to
centrations ranging from 0.01–0.05%) were added to media
form septa because of incomplete or aberrant chromo- at the time of inoculation. Because hyphae treated with low
some segregation. The recent finding that the predicted concentrations of HU (#5 mm) tend to recover before the
formation of the first septum, a second dose of HU was added
sepB gene product displays similarity to an S. cerevisiae
at the same concentration 6 hr after inoculation.
protein involved in chromosomal DNA metabolism is
Strains containing TS mutations were incubated at either
consistent with this hypothesis (Harris and Hamer
43.58(restrictive temperature) or 398(semipermissive
temper-1995). ature). For strains unable to undergo nuclear division at 398,
The sepB gene product may directly promote septum semipermissive conditions were empirically determined as the
maximum temperature allowing completion of at least two
formation in addition to its functions in chromosomal
rounds of nuclear division (generally 35.5–37.58).
DNA metabolism. Alternatively, the general
accumula-Staining, microscopy, and measurements:Fixation of
sam-tion of DNA damage caused by sepB mutasam-tions may ples, staining with Calcofluor and Hoechst 33258, and
mount-trigger a regulatory response that inhibits septation. The ing of stained coverslips on glass slides were performed as second possibility has been investigated by testing the previously described (Harriset al. 1994). Slides were observed using an Olympus (Olympus Corp., Lake Success, NY) BMAX
ability of other perturbations of chromosomal DNA
me-fluorescent microscope and DPLANAPO340 and3100 (oil
tabolism to prevent septum formation. Results
pre-immersion) objectives. Photographs were taken with Kodak
sented here demonstrate that septation is inhibited by Technical-Pan film (Eastman Kodak, Rochester, NY) and
de-a vde-ariety of trede-atments cde-apde-able of producing sublethde-al veloped in Kodak HC-110 developer.
amounts of DNA damage. In addition, genetic evidence The septation index (SI) describes the percentage of cells
possessing at least one septum within a randomly selected
is provided that suggests that septation is triggered by
population of 200 cells. Cell size was determined by measuring
high levels of cdk activity in A. nidulans, and that the
hyphal length with a calibrated eyepiece micrometer. Since
ability of defects in chromosomal DNA metabolism to fungal hyphae grow solely at the tip, the length of a hypha
prevent septum formation is dependent upon inhibitory generally indicates the extent to which it has grown. Under
Tyr-15 phosphorylation of the cdk p34nimX. Moreover,
the conditions used in these experiments, all strains exhibited similar hyphal diameters (i.e., 2–3mm). Thus, hyphal length
additional genetic interactions are presented that show
represent a reasonable approximation of cell size.
that the DNA damage-induced inhibition of septation
The nonparametric Mann-Whitney test was used to
deter-is enforced by a DNA damage checkpoint response. mine if two strains differed significantly in cell length at the
Finally, we propose that the timing of septum formation time the first septum formed (Zar 1984). To use this test,
measurements were ranked and the U and U9 values
calcu-in predivisional hyphal cells is controlled by a branched
lated. These values take the number of measurements and
regulatory pathway.
the sum of the ranks into consideration. The larger of the two values was compared to the respective critical value. If this value was greater than or equal to the critical value, the MATERIALS AND METHODS null hypothesis (that no significant difference in cell size
ex-ists) was rejected.
Strains, media, and genetic manipulations:All strains used
in this study are described in Table 1. Media used were YGV (2% glucose, 0.5% yeast extract, 0.1% vitamin mix) and CM6
RESULTS supplements (1% glucose, 0.2% peptone, 0.1% yeast extract,
0.1% casamino acids, nitrate salts, trace elements, and 0.01% General perturbations of DNA metabolism inhibit vitamins, pH 6.5). Supplements, vitamin mix, trace elements,
septum formation:The inability of sepB mutants to
un-and nitrate salts are described in the appendix to Kafer
dergo septation could be because of the accumulation
(1977). For solid media, 1.5% agar was added. Hydroxyurea
(HU; Sigma Chemical Co., St. Louis) was prepared as a 2 M of DNA damage resulting from a defect in chromosomal
stock solution and added to media after autoclaving. DNA metabolism (Harris and Hamer 1995). If this For the viability/aneuploidy experiment, conidia were di- notion is correct, then additional perturbations of chro-luted and plated for single colonies on CM plates. Plates were
mosomal DNA metabolism might also be expected to
incubated at 428for the indicated time period and then shifted
inhibit or delay septum formation. Among the sep
mu-to 288. Control plates were incubated at 288for the duration
TABLE 1
Aspergillus nidulans strains
Strain Genotype Sourcea
ASH15 sepB3; wA2; argB2 Lab stock
ASH35 sepA1 yA2; argB2 Lab stock
ASH201 uvsB110; chaA1b ASH153A832
ASH202 sepB3, uvsB110b ASH153A832
ASH203 sepB3; uvsB110b ASH153A832
ASH206 uvsD153; wA2b ASH153A571
ASH207 sepB3 uvsD153; wA2 ASH153A571
ASH247 sepJ1 wA2; argB2 Lab stock
ASH280 sepB3; nimXcdc2AF, wA2b ASH153FRY20
ASH281 sepB3; nimXcdc2AF, wA2b ASH153FRY20
ASH284 sepB3; wA2b ASH153FRY20
ASH286 nimXcdc2AF; wA2b ASH153FRY20
ASH299 sepA1; nimXcdc2AF; yA2b ASH353FRY20
ASH300 sepA1; yA2b ASH353FRY20
ASH326 sepJ1 nimXcdc2AFwA2b ASH2473FRY20
ASH329 sepJ1 wA2b ASH2473FRY20
AJM15 sepB4, wA2 Lab stock
A28 pabaA6; biA1 1
A571 uvsB153; pabaA125 biA1; fwA2 1
A777 bimC4, lacA1; adE20, biA1; wA2, cnxE16;
sC12; methG1; nicA2; choA1; chaA1 1
A781 nimA5; wA2 1
A832 uvsB110; pabaA1; nicA2; chaA1 1
SWJ010 bimE7; pabaA1 yA2 2
SWJ238 nimO18; riboA1; yA2 3
SWJ280 nimP22; pyroA4; chaA1 3
SWJ310 nimR21; pabaA1 3
SO65 nimX3Y306H; wA3; pyroA4; riboA1; yA2 4
SO75 nimE6; riboA1; yA2 4
FRY20 nimXcdc2AFwA3; pyrG89 ; pyroA4; pyr41 4
MO73 nimT23 wA2 4
JD139b nimL25; chaA1b 5
M3127 bimB3; yA2; wA3; proA1; pyrG89 6
aKey to sources: 1. Fungal Genetic Stock Center, Department of Microbiology, University of Kansas Medical Center, Lawrence, KS; 2. Dr. Peter Mirabito, University of Kentucky, Lexington, KY; 3. Dr. Steven James, Gettysburg College, Gettysburg, PA; 4. Dr. Stephen Osmani, Geisinger Clinic, Danville, PA; 5. Dr. John Doonan, John Innes Center, Norwich, UK; 6. Dr. Greg May, Baylor College of Medicine, Houston, TX.
bStrain may possess other uncharacterized markers.
closely resemble those observed in sepB mutants. In addi- To further test the notion that defects in
chromo-somal DNA metabolism lead to inhibition of septum tion to the defect in septum formation (Figure 1B; Table
2), plating experiments showed that sepJ1 mutants be- formation, the effects on septation of the replication
inhibitor HU and the DNA-damaging agent DEO were have like sepB mutants in rapidly losing viability
follow-ing prolonged incubation at restrictive temperature examined in wild-type cells. Specifically, conidia were
allowed to germinate and form hyphae in the presence (Figure 1A). Furthermore, the increase in proportion
of aneuploid survivors (Figure 1A) suggests that the of sublethal doses of these agents. Under such
condi-tions, low levels of DNA damage would presumably accu-sepJ1 mutation causes defects that lead to enhanced
chromosome loss and/or mitotic recombination mulate while growth and nuclear division would be only
modestly affected. Indeed, wild-type cells incubated in (Kafer and Upshall 1973). When examined
micro-scopically, sepJ1 mutants were found to possess morpho- the presence of 5 mm HU or 0.025% DEO for 11 hr
exceeded the size at which septation normally occurs logically aberrant nuclei whose appearance deteriorated
with each successive round of nuclear division (Figure (i.e.,$50mm,Harriset al. 1994; average cell sizes for
HU-treated and DEO-treated cells were 122.1 6 57.9
1, C–F). Collectively, these phenotypes suggest that the
sepJ1 mutation causes defects in chromosomal DNA me- mm and 1576 57.9 mm, respectively) and underwent
Figure 1.—The sepJ1 mutation prevents septum formation and also causes defects in chromosomal DNA metabolism. (A) Conidia from strains A28 (wild type, triangles), ASH329 (sepJ1, circles), and AJM15 (sepB4, squares) were diluted and plated for single colonies on CM plates. Plates were incubated at 428for the indicated time period and then shifted to 288. The zero time point was held at 288for the duration of the experiment. Open symbols represent the percentage of viable colonies relative to the zero time point. Closed symbols represent the percentage of viable colonies with abnormal morphologies characteristic of aneuploids (KaferandUpshall1973). (B) Wild-type (open circles) and sepJ1 (closed circles) hyphal cells were incubated on coverslips for 9 hr (wild type) or 14 hr (sepJ1) at 428. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. For each strain, the length and nuclear number of 25 randomly selected cells was measured and plotted as a scatter graph. In addition, the septation index (SI, the percentage of cells that possess at least one septum) was determined for a randomly selected population of 200 cells. For wild-type cells, the SI was 64%, whereas it was zero for sepJ cells. The strains analyzed were A28 (wild type) and ASH329 (sepJ1). (C–E) sepJ1 hyphal cells from this experiment. (F) Wild-type control. Arrow denotes a septum. Bar, 4mm.
cells and 98% of DEO-treated cells possessed $8 nu- cycle progression have been identified and
character-ized in A. nidulans (Morris 1976; Morris and Enos
clei). However, as predicted, septum formation was
sig-nificantly delayed in these cells (Figure 2). For example, 1992;Doonan1992). These mutants fail to form septa
at restrictive temperature because of their inability to in the population of cells possessing 16 nuclei, the
num-ber of untreated cells that had undergone septation was complete a single mitotic division (Morris 1976). In
contrast, because they undergo mitosis and are gener-approximately eightfold greater than the number of
treated cells (Figure 2A). Moreover, at this stage, un- ally not impaired for growth, most of these mutants are
expected to form septa at semipermissive temperatures.
treated cells were much shorter (56.8618.1mm) than
the treated cells (Figure 2, B–D). However, if a specific TS mutation causes defects in
TABLE 2 tent with the notion that low levels of DNA damage inhibit septation without blocking nuclear division.
Septum formation in sep uvs and sep nimXcdc2AF
Septum formation requires high levels of p34nimX activ-double mutants
ity:In A. nidulans, the cdk encoded by the nimX gene
(p34nimX) is required for progression through the
nu-Strain Genotype Septation index (%)
clear division cycle (Osmani et al. 1994). The nimX3
A28 wild type 87.5
mutation confers cell cycle arrest in both G1and G2with
ASH284 sepB3 1.0
severely reduced kinase activity (Osmaniet al. 1994;Ye
ASH329 sepJ1 1.5
et al. 1995). When the ability of the TS cell cycle mutants
ASH300 sepA1 0
to undergo septation at semipermissive temperature was
ASH286 nimXcdc2AF 79.0
ASH201 uvsB110 86.5 examined, it was observed that the nimX3 mutation
pre-ASH206 uvsD153 93.5 vented septum formation (Figure 3D). Although the
ASH280 sepB3 nimXcdc2AF 64.0
majority of nimX3 cells had exceeded the size at which
ASH203 sepB3 uvsB110 78.5 wild-type cells undergo septation ($50mm) and
pos-ASH207 sepB3 uvsD153 82.5
sessed four or more nuclei (Figure 3, A and D), they
ASH326 sepJ1 nimXcdc2AF 59.5
yielded an SI of only 1.5%. Furthermore, nimX3 hyphae,
ASH299 sepA1 nimXcdc2AF 0
which did form at least one septum, were .150 mm
Conidia were germinated on coverslips for 12–15 hr at 428.
in length and possessed .8 nuclei (n 5 10). These
Coverslips were stained with Calcofluor and Hoechst 33258
observations suggest that p34nimXactivity is required for to visualize septa and nuclei, respectively. The septation index
septation in A. nidulans.
represents the percentage of cells which possess at least one
septum (n5200). The A. nidulans nimT gene encodes the tyrosine
phos-phatase which triggers cdk activation by dephosphory-lating the Tyr-15 residue of p34nimX (O’Connell et al.
1992). At restrictive temperature, nimT23 mutants arrest
hibited or severely delayed under these conditions. in G2with low levels of p34nimXhistone H1 kinase activity
Among this collection of mutants, cells possessing the (Yeet al. 1995). If p34nimXactivity is required for septation
nimL15 mutation display a variety of phenotypes consis- in A. nidulans, then the nimT23 mutation should affect
tent with a defect in chromosomal DNA metabolism. At septum formation in a manner similar to that observed
restrictive temperature, nimL15 mutants fail to undergo for the nimX3 mutation. Indeed, the nimT23 mutation
nuclear division and appear to arrest in S phase (Morris
caused a severe delay in septum formation at
semiper-1976;Doonan1992). Furthermore, at both permissive missive temperature (Figure 4, A and B; note that Figure
and semipermissive temperatures, strains possessing this 4 presents data from cells possessing one septum,
mutation are sensitive to HU and methyl methanesulfo- whereas Figure 3 presents data from a random
popula-nate (MMS) and also exhibit enhanced levels of sponta- tion of cells). The majority of nimT23 hyphae did not
neous mutagenesis (Doonan1992;S. Harris, unpub- undergo septation until they were$150 mm long and
lished results). When incubated at semipermissive had accumulated more than eight nuclei. These results
temperature, nimL15 mutants grow to an adequate size are consistent with the notion that cdk activity is
re-(i.e., .50mm long) and undergo sufficient rounds of quired for septation in A. nidulans. Furthermore, the
nuclear division for septation to occur (Figure 3, A and observation that nim X3 and nimT23 mutants are capable
B). Nonetheless, the SI for nimL15 mutants was only of completing multiple rounds of nuclear division at
4%, and those cells possessing septa were$100mm long semipermissive temperature suggests that the level of
and possessed$14 nuclei. Similar results were obtained p34nimXactivity needed to trigger septation may be higher
when cells possessing the nimP22 mutation were exam- than that required for mitosis.
ined for septum formation following incubation at semi- The inability to phosphorylate the Tyr-15 residue of
permissive temperature (Figure 3C). The nimP gene p34nimX leads to elevated levels of histone H1 kinase
encodes the A. nidulans homologue of DNA polymerase activity and causes defects in checkpoint regulation (Ye
ε(S. James,personal communication), which is thought et al. 1996, 1997;OsmaniandYe 1997). If cdk activity
to be required for both chromosomal DNA replication plays a critical role in the temporal control of septum
and for replication-mediated DNA repair (Stillman formation, then the increase in p34nimXactivity observed
1994). In contrast, most of the remaining cell cycle when the Tyr-15 residue cannot be phosphorylated may
mutants underwent septation with kinetics, which did also promote premature septation. The nimXcdc2AFallele
not differ significantly from wild type. Thus, a diverse encodes a p34nimX, which is no longer subject to
inhibi-array of mutational (i.e., sepB3, sepB4, sepJ1, nimL15, tory phosphorylation at Tyr-15 (Yeet al. 1996). As
pre-nimP22) and pharmacological (sublethal doses of HU or dicted, hyphae possessing this mutation generally
un-DEO) perturbations of chromosomal DNA metabolism derwent septation at a smaller size and with fewer nuclei
share the common effect of severely delaying or inhib- compared to wild-type cells (Figure 4, A and C). For
Figure 3.—Mutations that cause defects in chromosomal
Figure 4.—Septum formation requires high levels of mi-DNA metabolism also prevent septum formation. Hyphal cells
totic cdk activity. Hyphal cells of the indicated genotypes were of the indicated genotypes were incubated on coverslips for
incubated on coverslips for 9 hr (wild type) or 12 hr (nim 9 hr (wild type) or 14 hr (nim mutants) at 378. Coverslips were
mutants) at either 378(wild type, nimXcdc2AF, and nimE6) or stained with Calcofluor and Hoechst 33258 to visualize septa
398 (nimT23). Coverslips were stained with Calcofluor and and nuclei, respectively. For each strain, the length and
nu-Hoechst 33258 to visualize septa and nuclei, respectively. For clear number of 25 randomly selected cells was measured and each strain, the length and nuclear number of 25 cells pos-plotted as a scatter graph. In addition, the SI was determined sessing one septum was measured and plotted as a scatter for a randomly selected population of 200 cells. The strains graph. In addition, the SI was determined for a randomly analyzed were: (A) A28, (B) JD139, (C) SWJ280, and (D) selected population of 200 cells. The following strains were
SO65. analyzed: (A) A28, (B) MO73, (C) FRY 20, and (D) SO75.
#50mm long when septum formation occurred, 52% in A. nidulans hyphae is temporally controlled by cdk
of nimXcdc2AF cells underwent septation within this size activity. To date, the only cyclin identified in A. nidulans
range. In addition, 24% of nimXcdc2AF cells contained is the B-type cyclin encoded by the nimE gene (
O’Con-four or fewer nuclei when the first septum formed, a nellet al. 1992). Association of this cyclin with p34nimX
condition never observed in wild-type cells (Harriset is required for progression through both G1 and G2
al. 1994). Septum formation appears to be generally (O’Connell et al. 1992; Dayton et al. 1997). If the
deregulated in nimXcdc2AFmutants (Figures 5 and 7C). NimE/p34nimXcomplex is also required for septum
for-Small cells (#25 mm) containing one or more septa mation, loss of function mutations in the nimE gene
are observed at low frequency (7%), and a significant might be expected to delay or inhibit septation. In
con-fraction of hyphae were found to contain nuclei bisected trast, if a distinct cyclin is required for septum
forma-by a septum (16%, n5100). Similar effects upon septa- tion, nimE mutations would presumably have no effect
tion were also observed when nimA5 mutants were incu- upon septation. Incubation of cells possessing the nimE6
bated at semipermissive temperature (Wolkow et al. mutation at semipermissive temperature resulted in
de-1996). At restrictive temperature, the nimA5 mutation layed septation (Figure 4D). Despite completing two to
confers a G2 arrest in which p34nimX accumulates in a three rounds of nuclear division, hyphae formed by
Tyr-15 dephosphorylated and fully active state (Osmani nimE6 mutants were typically twice as long as wild-type
et al. 1991). Taken together, these observations suggest cells when the first septum formed. Although these
re-that elevated levels of p34nimXactivity trigger premature sults do not eliminate the possibility that additional cdk
formation of the first septum in A. nidulans hyphae. complexes promote septation, they do demonstrate that
The opposing effects of the nimT23 and nimXcdc2AF the same cdk complex that regulates mitosis also
con-mutations on the timing of septum formation provide trols septum formation.
gies at restrictive temperature (Figures 6 and 7D). Furthermore, at permissive temperature, they exhibited slower growth than either of the parental single mutants and were also sensitive to HU (Figure 6). However, despite the presence of the sepB3 mutation, the double mutants were able to undergo septation at restric-tive temperature (Figure 7, A–D; Table 2). Eight sepB3 nimXcdc2AFdouble mutant isolates derived from two
inde-pendent crosses were examined, and all were able to form septa at restrictive temperature. Two additional observations indicate that the ability of the nimXcdc2AF
mutation to permit septation in the presence of suble-thal DNA damage is not specific to the defects caused by the sepB3 mutation. First, sepJ1 nimXcdc2AFdouble mutants Figure5.—Deregulation of septum formation in nimXcdc2AF
undergo septation at restrictive temperature (Figure 7,
mutants. Conidia of strain FRY 20 were germinated on
cov-E and F; Table 2), and second, cells possessing the
erslips for 9 hr at 378. Coverslips were stained with Calcofluor
and Hoechst 33258 to visualize septa and nuclei, respectively. nimXcdc2AFmutation were able to form septa when grown Cell 1 is binucleate and possesses a septum. Cell 2 has formed in the presence of 0.025% DEO (i.e., when incubated a double septum. Cell 3 possesses an anucleate compartment. in DEO, 54% of cells possessing eight nuclei had under-Bar, 4mm.
gone septation, compared to 61% for untreated cells). The possibility that these epistatic interactions reflect the ability of the nimXcdc2AF mutation to circumvent all septation when chromosomal DNA metabolism is
per-of the controls which regulate septum formation was
turbed:Since septum formation is temporally regulated
eliminated by showing that sepA1 nimXcdc2AFdouble
mu-by p34nimXactivity, defects in chromosomal DNA
metabo-tants do not undergo septation at restrictive tempera-lism could conceivably inhibit septation by reducing the
ture (Table 2). The sepA1 mutation confers a late-acting level of active kinase. One mechanism by which this
defect in septum formation (Harriset al. 1994), and
could be accomplished is through increased Tyr-15
the sepA gene product is thought to be required for the phosphorylation of p34nimX
. To test this notion, a sepB3
organization of the actin ring at the division site (
Har-nimXcdc2AFdouble mutant was constructed and tested for
riset al. 1997). Collectively, these results show that
in-the ability to undergo septation at restrictive
tempera-hibitory Tyr-15 phosphorylation of p34nimX is required
ture. The presence of septa in the double mutant would
to prevent septation in sepB3 and sepJ1 mutants, and
indicate that the effects of p34nimX on septation were
suggest that defects in chromosomal DNA metabolism epistatic to those of the sepB3 mutation. In contrast, the
may prevent septum formation by modulating cdk ac-absence of septa would suggest that defects in
chromo-tivity. somal DNA metabolism influence septation at a point
The inability of sepB3 mutants to form septa requires
concomitant with or downstream of p34nimX.
a functional DNA damage checkpoint pathway:Normal sepB3 nimXcdc2AFdouble mutants were found to be TS
for growth and to display aberrant nuclear morpholo- function of the DNA damage checkpoint in A. nidulans
Figure7.—Cellular morphology of the double mutants. Conidia were germinated on coverslips for 9–15 hr at 428. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. (A) A28 (wild type), (B) ASH284 (sepB3), (C) ASH286 (nimXcdc2AF), (D) ASH280 (sepB3 nimXcdc2AF), (E) ASH329 (sepJ1), (F) ASH326 (sepJ1 nimXcdc2AF), (G) ASH203 (sepB3 uvsB110), and (H) ASH207 (sepB3 uvsD153). Arrows denote septa. Bar, 4mm.
requires inhibitory Tyr-15 phosphorylation of p34nimX require other components of the DNA damage
check-point. To examine this possibility, sepB3 uvsB110 and (Yeet al. 1997). Since this mechanism also acts to
pre-vent septum formation when chromosomal DNA metab- sepB3 uvsD153 double mutants were constructed and
tested for their ability to undergo septation at restrictive olism has been perturbed, it seemed possible that the
mu-TABLE 3 contrast, septum formation should occur at a smaller than normal size in cells possessing both the sepB3 Septum formation in checkpoint mutants treated with a
and nimXcdc2AFmutations. We found that sepB3 uvsB110
DNA-damaging agent
double mutants were significantly longer than sepB3 nimXcdc2AFmutants when they underwent septation (i.e., Septation index (%)
100.26 40.9 mm vs. 34.8 610.3 mm,a 5 0.001, Z5
Strain Genotype Control 1DEO
6.05). Similar results were obtained for the analogous
A28 wild type 93.0 19.0 comparison involving sepB3 uvsD153 double mutants
A832 uvsB110 99.0 58.0 (i.e., 109.0 6 42.0mm vs. 34.86 10.3mm, a 50.001,
A571 uvsD153 94.0 51.0 Z 5 6.01). These results suggest that the inability to
phosphorylate the Tyr-15 residue of p34nimX abolishes
Conidia were germinated on coverslips for 12 hr at 288.
the regulatory mechanisms that: (i) prevent septum
for-Coverslips were stained with Calcofluor and Hoechst 33258
to visualize septa and nuclei, respectively. The septation index mation until cells have grown to the appropriate size,
represents the percentage of cells which possess at least one and (ii) inhibit septation in the presence of DNA dam-septum (n5200).
age. In contrast, the uvsB and uvsD gene products regu-late septation only when DNA damage has been in-curred. Thus, the regulatory pathway that controls the tants suggests that mutations in these genes abrogate
timing of septation must be branched, with different
the DNA damage checkpoint (Ye et al. 1997).
Specifi-gene products controlling the responses to cell size and
cally, uvsB and uvsD mutants are sensitive to UV irradia- DNA damage.
tion, MMS, and low levels of HU (Kaferand Mayor
1986; Figure 6). In addition, they fail to delay entry into
DISCUSSION
mitosis when incubated in the presence of 0.01% MMS (P. KrausandS. Harris,unpublished results).
Germinating A. nidulans conidia complete multiple
sepB3 uvsB110 and sepB3 uvsD153 double mutants rounds of nuclear division and form an elongated
hy-were TS for growth and displayed aberrant nuclear mor- phal cell before the formation of the first septum (
Har-phologies at restrictive temperature (Figure 6; Figure riset al. 1994;Wolkow et al. 1996). Previous analyses
7, G and H). Furthermore, they exhibited a synthetic of a specific class of septation mutants suggested that a
slow growth phenotype as well as sensitivity to HU at specific regulatory mechanism prevents septation when
permissive temperature (Figure 6). However, despite the fidelity of the early rounds of nuclear division is
the presence of the sepB3 mutation, the double mutants compromised (Harriset al. 1994;HarrisandHamer
were able to form septa at restrictive temperature (Fig- 1995). Here, the regulatory circuit that controls the
ure 7, G and H; Table 2). These observations suggest timing of septum formation in predivisional hyphal cells
that a functional DNA damage checkpoint is required is characterized in greater detail. In particular, it is
to prevent septum formation in sepB3 mutants. More- shown that: (i) general defects in chromosomal DNA
over, this effect is not specific to the defects in chromo- metabolism inhibit the formation of the first septum,
somal DNA metabolism caused by the sepB3 mutation, (ii) inhibition of septum formation requires an intact
because uvsB110 and uvsD153 mutants are also able to DNA damage checkpoint pathway, and (iii) distinct
sen-form septa in the presence of 0.025% DEO (Table 3). sory inputs control the timing of septation by
modulat-A branched pathway regulates the timing of septum ing the activity of an interphase cdk complex. formation:Because cells possessing the nimXcdc2AF
muta-General defects in chromosomal DNA metabolism
tion alone undergo “premature septation” (i.e., forma- prevent septum formation in predivisional hyphal cells:
tion of the first septum at a small size and with fewer Phenotypic characterization of the TS sepB3 and sepB4
nuclei compared to wild-type cells, Figures 4C and 5), mutations raised the possibility that the accumulation
the ability of this mutation to permit septum formation of DNA damage may inhibit formation of the first
sep-in sepB3 mutants presumably reflects the bypass of most tum in germinating A. nidulans conidia (Harrisand
of the controls that temporally regulate this process. In Hamer1995). Conceivably, this response could be
trig-contrast, the uvsB110 mutation by itself does not affect gered solely by DNA damage generated as a result of
the timing of septation. The majority of cells possessing sepB mutations. However, the observation that a variety
this mutation were between 50 and 75mm in length and of mutational (i.e., incubation of sepJ1 mutants at
restric-contained .8 nuclei when they underwent septation. tive temperature and nimL15 or nimP22 mutants at
semi-Thus, whereas the regulatory pathway that prevents sep- permissive temperatures) and pharmacological (i.e.,
tum formation in the presence of DNA damage has treatment of wild-type cells with sublethal doses of HU
been disabled in uvsB110 mutants, the cell size control or DEO) perturbations of chromosomal DNA
metabo-remains functional. If this notion is correct, sepB3 lism inhibit or delay septum formation suggests that this
uvsB110 double mutants should undergo septation is a general response activated by multiple types of DNA
damage. In support of this notion, the genotoxic effects
of low doses of HU or DEO are likely to be very different. yeast (Enoch and Norbury 1995), these phenotypes
suggest that the uvsB and uvsD gene products are re-Treatment with HU causes depletion of nucleoside
tri-phosphate pools and presumably leads to the accumula- quired for a signal transduction component, which
un-derlies both checkpoint responses. The ultimate
ef-tion of base substituef-tions (Kunzand Kohalmi 1991).
In contrast, the high frequency of deletions induced by fector of the DNA damage checkpoint in A. nidulans is
the mitotic cdk p34nimX (Ye et al. 1997). Whereas the
DEO in fungal cells indicates that it causes DNA strand
breaks (Ongand De Serres1975; Hynes1979). Fur- nimXcdc2AFmutation abrogates the DNA damage
check-point response and permits mitotic entry in UV-irradi-thermore, preliminary results suggest that the effects of
sepB and nimL mutations on chromosomal DNA metabo- ated or MMS-treated cells (Yeet al. 1997), it does not
affect the checkpoint that responds to the arrest of DNA lism also differ significantly. Unlike mutations in sepB,
at semipermissive temperatures, the nimL15 mutation replication (Yeet al. 1996). The later response also
re-quires inactivation of the bimE gene product (Ye et al.
causes sensitivity to both HU and MMS and promotes
increased levels of spontaneous mutagenesis (S. Harris, 1996), a negative regulator of mitosis, which is part of
the anaphase-promoting complex (James et al. 1995;
unpublished observations).
It is important to note that the DNA damage that OsmaniandYe1997).
The nimXcdc2AFmutation, as well as mutations in uvsB
prevents septum formation in predivisional hyphal cells
does not block nuclear division. Each of the sep and nim or uvsD, permits inappropriate septum formation in
predivisional hyphal cells that have incurred DNA dam-mutants, as well as wild-type cells treated with sublethal
doses of HU or DEO, typically complete at least three age. Notably, each of these mutations exacerbates many
of the phenotypes caused by the sepB3 mutation. For rounds of nuclear division following the imposition of
damage-inducing conditions. Although the kinetics of example, nimXcdc2AFsepB3 and uvs sepB3 double mutants
display severe defects in nuclear morphology and grow nuclear division have not been systematically examined
under these conditions, the sepB3 mutation appears to slowly even at permissive temperature. Thus the ability
of these double mutants to undergo septation does not
cause only a slight delay in mitosis (HarrisandHamer
1995). Two possible explanations could account for the reflect a general suppression of the defects in
chromo-somal DNA metabolism induced by the sepB3 mutation. differential effects of DNA damage on septum
forma-tion and nuclear division in germinating conidia. One Instead, these observations demonstrate that the DNA
damage-induced inhibition of septum formation in pre-possibility is that septation is specifically inhibited by a
certain type of damage that does not affect mitosis. divisional hyphal cells is enforced by a checkpoint
re-sponse. However, the ability of a broad range of
damage-induc-ing conditions to prevent septum formation casts doubt Distinct sensory inputs control the timing of septum
formation by modulating the activity of an interphase
upon this notion. Alternatively, the response to DNA
damage in predivisional cells may be quantitative. In cdk complex: The phenotypes displayed by
predivi-sional hyphal cells possessing the nimXcdc2AF mutation
particular, the threshold of damage required to prevent
septation may be lower than that needed to inhibit nu- clearly indicate that most of the controls that regulate
the timing of septum formation have been abrogated. clear division. This idea is supported by the observation
that nuclear division is inhibited by doses of HU (>20 These include the controls that: (i) prevent septum
formation in the presence of DNA damage, and (ii) mM) or DEO (>0.05%) higher than those which
pre-vent septation. Moreover, the epre-ventual failure of nuclear establish the appropriate cell size at which septation
occurs. In contrast, the uvsB and uvsD mutations only division in sepB and sepJ mutants may be caused by the
gradual accumulation of irreparable DNA damage until appear to abolish the DNA damage response (i.e., uvs
sepB double mutants do not septate at small cell size). levels exceed the threshold that prevents cell cycle
pro-gression. These observations suggest that the formation of the
first septum in germinating conidia is temporally
con-Inhibition of septum formation in predivisional
hy-phal cells requires an intact DNA damage checkpoint: trolled by distinct sensory inputs that control the re-sponses to DNA damage and cellular growth (Figure 8). The initial characterization of both the S-phase and
DNA damage checkpoint responses in A. nidulans has Furthermore, these signals converge on the regulatory
module which controls Tyr-15 phosphorylation of recently been reported (Ye et al. 1996;Ye et al. 1997).
The phenotypes displayed by uvsB and uvsD mutants p34nimX (LewandKornbluth 1996). This module
in-cludes the Wee1 tyrosine kinase and the Cdc25 tyrosine suggest that these gene products function in each of
these checkpoint responses (Ye et al. 1997). Mutations phosphatase, which are encoded by the ankA and nimT
genes, respectively, in A. nidulans (O’Connell et al.
in either of these genes confers sensitivity to UV
irradia-tion, MMS, and HU (KaferandMayor 1986; Figure 1992; Ye et al. 1997). Regulatory signals which act to
delay septum formation presumably lower the levels of 4). Furthermore, uvsB and uvsD mutants fail to delay
mitotic entry when treated with MMS (P. KrausandS. active p34nimXby increasing ankAwee1activity and/or
de-creasing nimTcdc25activity. This response could conceiv-Harris,unpublished results). By analogy to the
interphase cdk activity to levels below the threshold needed for septation. Additional experiments designed to assess the effects of sepB and sepJ mutations on the
Tyr-15 phosphorylation state and activity of p34nimX
should test the validity of this notion.
The timing of septum formation and its role in hyphal differentiation: A. nidulans postdivisional hyphae are
differentiated in the sense that they possess two distinct
Figure 8.—A branched regulatory pathway controls the cell types (Fiddy and Trinci 1976; Harris 1997; timing of septum formation in predivisional hyphal cells. Dis- Kaminskyj and Hamer 1998): (i) a tip cell in which tinct sensory inputs control the responses to DNA damage
cell cycle progression and polarized hyphal extension
and cell growth. The damage response requires elements of
continue unabated, and (ii) subapical cells, which enter
the DNA damage checkpoint pathway (uvsB and uvsD), and
a period of mitotic quiescence and growth arrest.
Sub-is activated by a variety of defects in chromosomal DNA
metab-olism (including those caused by mutation in sepB and sepJ ). apical cells are likely to serve two important functions
The response to growth conditions (i.e., cell size) remains in hyphae. First, they function as “stem cells” for the uncharacterized. Both sensory inputs presumably converge
formation of asexual and sexual reproductive structures
on the regulatory module which controls the activity of the
(Timberlake1990). Second, the presence of nondivid-mitotic cdk p34nimX. Activated p34nimX is predicted to trigger
ing nuclei in these cells appears to provide
postdivi-septation by modifying a gene product which is required for
the activation of the late-acting sep genes. Double mutant sional hyphae with some degree of resistance to the
analyses indicate that the sepA gene product functions up- lethal effects of DNA damage. Indeed, postdivisional stream of sepD, sepG, and sepH (Morrell1997).
hyphae are significantly less sensitive to the effects of both UV irradiation and loss of sepB function than are
predivisional hyphal cells (S. Harris,unpublished
ob-(O’Connellet al. 1997;Furnari et al. 1997;Sanchez
servations). For these reasons, it is undoubtedly crucial
et al. 1997). Because the ultimate effect of the regulatory that nuclei within subapical cells possess chromosomes
pathway is the inhibition of septum formation, p34nimX
that are intact and undamaged. Thus, the importance
must interact with a substrate that is required for septa- of the DNA damage-induced inhibition of septation in
tion. Given that sepA1 nimXcdc2AF double mutants fail to
predivisional hyphal cells may lie in its ability to prevent
septate, and that SepA is required for actin ring forma- cellularization if unrepaired chromosomes enter mitosis
tion at the division site (Harriset al. 1997), potential
(because of failed repair or adaptation to the
check-substrates include SepA or the gene products that regu- point signal,Paulovichet al. 1997). In such a context,
late its activity. this response may represent an important
develop-Results presented here show that p34nimXis required
mental checkpoint in A. nidulans. for both mitosis and septation in predivisional hyphal
We thankJohn Doonan, Steven James, Greg May, Peter
Mira-cells. Moreover, the ability of these cells to undergo bito, Stephen Osmani, and Xiang Ye for generously providing
mitosis in the absence of septum formation implies that strains;Jennifer Gittzusfor providing the data depicted in Table
these effects are separable. The existence of such a 3; Xiang Ye, Stephen Osmani,and Tom Wolkow for insightful
discussion and the communication of unpublished results; andJohn
relationship would presumably be undetectable in
uni-Hamerfor a critical evaluation of the manuscript. This work was
nucleate cells, where it would be masked by the
obliga-supported by a grant from the National Science Foundation
(MCB-tory coupling of cytokinesis to mitosis (Satterwhite
9513489).
andPollard1992). One potential explanation for the
different effects of p34nimXactivity on mitosis and
septa-tion is that there exists a “septasepta-tion-specific” cyclin
sub-LITERATURE CITED
unit. However, the observation that mutations in the
NimE cyclin delay septum formation does not support Andreassen, P. R., D. K. Palmer, M. H. WenerandR. L.
Margo-lis,1991 Telophase disc: a new mammalian mitotic organelle
this hypothesis. Instead, the data are consistent with a
that bisects telophase cells with a possible function in cytokinesis.
model whereby septation requires attainment of thresh- J. Cell Sci. 99: 523–534.
old levels of interphase p34nimX activity. In particular,
Cooke, C. A., M. S. HeckandW. C. Earnshaw,1987 The inner
centromere protein (INCENP) antigens: movement from inner
levels of kinase activity may be assessed at a specific
centromere to midbody during mitosis. J. Cell Biol. 105: 2053–
point during interphase in predivisional hyphal cells. 2067.
Only if activity exceeds the threshold does septation Dayton, J., M. Sumi, N. NanthakumarandA. R. Means,1997
Ex-pression of a constitutively active Ca21/calmodulin-dependent
occur following the next round of mitosis. An important
kinase in Aspergillus nidulans spores prevents germination and
test of this model will be to compare cdk activity during entry into the cell cycle. J. Biol. Chem. 272: 3223–3230. mitoses that are followed by septation to those that are Doonan, J. H., 1992 Cell division in Aspergillus. J. Cell Sci. 103:
599–611.
not. Moreover, the model predicts that accumulation
Earnshaw, W. C., andA. M. Mackay, 1994 Role of nonhistone
of sublethal DNA damage inhibits septum formation by proteins in the chromosomal events of mitosis. FASEB J. 8: 947–
956.
Enoch, T.,andC. Norbury,1995 Cellular responses to DNA dam- a component of the spindle assembly checkpoint, required to
prevent septum formation and premature exit from mitosis if age: cell cycle checkpoints, apoptosis, and the roles of p53 and
ATM. Trends Biochem. Sci. 20: 426–430. spindle function is compromised. EMBO J. 15: 6605–6616.
O’Connell, M. J., A. H. Osmani, N. R. MorrisandS. A. Osmani, Fankhauser, C., J. Marks, A. ReymondandV. Simanis,1993 The
S. pombe cdc16 gene is required both for maintenance of p34cdc2 1992 An extra copy of nimEcyclinBelevates pre-MPF levels and partially suppresses mutation of nimTcdc25in Aspergillus nidulans. kinase activity and regulation of septum formation: a link between
mitosis and cytokinesis? EMBO J. 12: 2697–2704. EMBO J. 11: 2139–2149.
O’Connell, M. J., J. M. Raleigh,H. M. VerkadeandP. Nurse, Fiddy, C.,andA. P. J. Trinci,1976 Mitosis, septation, branching
and the duplication cycle in Aspergillus nidulans. J. Gen. Microbiol. 1997 Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J. 16: 545–554.
97:169–184.
Field, C. M.,andB. M. Alberts,1995 Anillin, a contractile ring Oegema, K.,andT. J. Mitchison,1997 Rappaport rules: cleavage
furrow induction in animal cells. Proc. Natl. Acad. Sci. USA 94: protein that cycles from the nucleus to the cell cortex. J. Cell
Biol. 131: 165–178. 4817–4820.
Ong, T.-M.,andF. J. De Serres,1975 Mutation induction by difunc-Fishkind, D. J.,andY.-L. Wang,1995 New horizons for cytokinesis.
Curr. Opin. Cell Biol. 7: 23–31. tional alkylating agents in Neurospora crassa. Genetics 80: 475–482.
Osmani, S. A.,andX. S. Ye,1997 Targets of checkpoints controlling Furnari, B., N. RhindandP. Russell,1997 Cdc25 mitotic inducer
targeted by Chk1 DNA damage checkpoint kinase. Science 277: mitosis: lessons from lower eukaryotes. Trends Cell Biol. 7: 283– 288.
1495–1497.
Harris, S. D.,1997 The duplication cycle in Aspergillus nidulans. Osmani, A. H.,S. L. McGuire andS. A. Osmani, 1991 Parallel
activation of the NIMA and p34cdc2cell cycle-regulated protein Fungal Genet. Biol. 22: 1–12.
Harris, S. D.,andJ. E. Hamer,1995 sepB: an Aspergillus nidulans kinases is required to initiate mitosis in A. nidulans. Cell 67:
283–291. gene involved in chromosome segregation and the initiation of
cytokinesis. EMBO J. 14: 5244 –5257. Osmani, A. H., N. vanPeij, M. Mischke, M. J. O’Connell and S. A. Osmani,1994 A single p34cdc2protein kinase (encoded by Harris, S. D., J. L. MorrellandJ. E. Hamer,1994 Identification
and characterization of Aspergillus nidulans mutants defective in nimXcdc2) is required at G1and G2in Aspergillus nidulans. J. Cell Sci. 107: 1519–1528.
cytokinesis. Genetics 136: 517–532.
Harris, S. D., L. Hamer, K. E. SharplessandJ. E. Hamer,1997 Paulovich, A. G., D. P. ToczyskiandL. H. Hartwell,1997 When
checkpoints fail. Cell 88: 315–321. The Aspergillus nidulans sepA gene encodes an FH1/2 protein
involved in cytokinesis and the maintenance of cellular polarity. Sanchez, Y., C. Wong, R. S. Thoma, R. Richman, Z. Wuet al., 1997
Conservation of the Chk1 checkpoint pathway in mammals: link-EMBO J. 16: 3474–3483.
Hynes, M. J.,1979 Fine-structure mapping of the acetamidase struc- age of DNA damage to Cdk regulation through Cdc25. Science
277:1497–1501. tural gene and its controlling region in Aspergillus nidulans.
Genet-ics 91: 381–392. Satterwhite, L. L.,andT. D. Pollard,1992 Cytokinesis. Curr.
Opin. Cell Biol. 4: 43–52.
James, S. W., P. M. Mirabito, P. C. ScacherandN. R. Morris,1995
The Aspergillus nidulans bimE (blocked-in-mitosis) gene encodes Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. J. Ciseket al., 1992 Phosphorylation of myosin-II regulatory light
multiple cell cycle functions involved in mitotic checkpoint
con-chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis. trol and mitosis. J. Cell Sci. 108: 3485–3499.
J. Cell Biol. 118: 595–605.
Kafer, E.,1977 Meiotic and mitotic recombination in Aspergillus
Stillman, B.,1994 Smart machines at the DNA replication fork.
and its chromosomal aberrations. Adv. Genet. 19: 33–131.
Cell 78: 725–728.
Kafer, E.,andO. Mayor,1986 Genetic analysis of DNA repair in
Timberlake, W. E.,1990 Molecular genetics of Aspergillus
develop-Aspergillus: evidence for different types of MMS-sensitive
hyper-ment. Annu. Rev. Genet. 24: 5–36. rec mutants. Mutat. Res. 161: 119–134.
Wheatley, S. P.,andY.-L. Wang,1996 Midzone microtubule bun-Kafer, E.,andA. Upshall,1973 The phenotypes of the eight
diso-dles are continuously required for cytokinesis in cultured epithe-mics and trisoepithe-mics of Aspergillus nidulans. J. Hered. 64: 35–38.
lial cells. J. Cell Biol. 135: 981–989.
Kaminskyj, S. G. W.,andJ. E. Hamer,1998 hyp loci control cell
Williams, B. C., M. F. Riedy, E. V. Williams, M. GattiandM. L.
pattern formation in the vegetative mycelium of Aspergillus
nidu-Goldberg,1995 The Drosophila kinesin-like protein KLP3A is
lans. Genetics 148: in press.
a midbody component required for central spindle assembly and
Kunz, B. A.,andS. E. Kohalmi,1991 Modulation of mutagenesis
initiation of cytokinesis. J. Cell Biol. 129: 709–723. by deoxyribonucleotide levels. Annu. Rev. Genet. 25: 339–359.
Wolkow, T. D., S. D. HarrisandJ. E. Hamer,1996 Cytokinesis in Lew, D. J.,andS. Kornbluth,1996 Regulatory roles of cyclin
depen-Aspergillus nidulans is controlled by cell size, nuclear positioning
dent kinase phosphorylation in cell cycle control. Curr. Opin.
and mitosis. J. Cell Sci. 109: 2179–2188. Cell Biol. 8: 795–804.
Yang, S. S., E. Yeh, E. D. SalmonandK. Bloom,1997 Identification Lew, D. J.,andS. I. Reed,1993 Morphogenesis in the yeast cell cycle:
of a mid-anaphase checkpoint in budding yeast. J. Cell Biol. 136: regulation by Cdc28 and cyclins. J. Cell Biol. 120: 1305–1320.
345–354.
Martineau, S. N., P.R. Andreassenand R. L. Margolis, 1995
Ye, X. S., G. Xu, R. T. Pu, R. R. Fincher, S. L. McGuireet al., 1995
Delay of HeLa cell cleavage into interphase using
dihydrocytocha-The NIMA protein kinase is hyperphosphorylated and activated lasin B: retention of a post-mitotic spindle and telophase disc
downstream of p34cdc2/cyclin B: coordination of two mitosis pro-correlates with synchronous cleavage recovery. J. Cell Biol. 131:
moting kinases. EMBO J. 14: 986–994.
191–205. Ye, X. S., R. R. Fincher, A. Tang, K. O’DonnellandS. A. Osmani,
Momany, M.,and J. E. Hamer,1997 The relationship of actin,
1996 Two S-phase checkpoint systems, one involving the func-microtubules, and crosswall synthesis during septation in Aspergil- tion of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit
lus nidulans. Cell Motil. Cytoskel. 38: 373–384. NIMA and prevent premature mitosis. EMBO J. 15: 3599–3610.
Morrell, J.,1997 A molecular genetic analysis of septation in
Asper-Ye, X. S., R. R. Fincher, A. Tangand S. A. Osmani, 1997 The
gillus nidulans. Ph.D. Thesis, Purdue University, West Lafayette, G2/M DNA damage checkpoint inhibits mitosis through Tyr15
IN. phosphorylation of p34cdc2in Aspergillus nidulans. EMBO J. 16:
Morris, N. R.,1976 Mitotic mutants of Aspergillus nidulans. Genet. 182–192.
Res. 26: 237–254. Zar, J. H., 1984 Biostatistical Analysis. Prentice-Hall, Englewood Morris, N. R.,andA. P. Enos,1992 Mitotic gold in a mold: Aspergil- Cliffs, NJ.
lus genetics and the biology of mitosis. Trends Genet. 8: 32–37.