MITOSIS
IN
THE
YEAST
C. F . R O B I N O W ,
LIPOMYCES
LIPOFER*
M.D.
From the Department of Bacteriology, University of Western Ontario, London, Canada
ABSTRACT
A study of mitosis in Lipomyces has been carried out because preliminary observations by
Ganesan and Roberts, 1959 (9), had indicated that the nucleus of this yeast might be unusually favourable for morphological observations. This impression has proved correct.
The chromosomes of Lipomyces are visible as separate, countable bodies for the greater part
of mitosis. The pattern of mitosis differs from the common one in that in Lipomyces the
proper distribution of sister chromosomes is accomplished without the help of a spindle
apparatus. At the end of prophase sister chromosomes are found in pairs which align themselves parallel to one another to form a palisade or stack whose long axis coincides with
the axis of the impending division. At anaphase-telophase the stack of paired chromosomes
fuses into a seemingly homogeneous cord which divides by constriction.
INTRODUCTION
The mode of divison of the nucleus of baker's
yeast (Saccharomyces) has been studied for many
years but remains difficult to understand despite
improvements in staining techniques and the
advent of electron microscopy. Chromosomes
have been convincingly demonstrated at meiosis
(21) but few workers claim to have been able to
follow the movements of the chromosomes in
dividing nuclei of cells multiplying by budding.
In fact, there is good evidence that the yeast
nucleus divides directly and that in the course of
this process its chromosomes do not normally
become visible in the light microscope as separate, countable individuals (10, 24, 25, 26). The
poor visibility of the nucleus in living yeast examined by ordinary microscopy (the excellent
photographs of budding cells by Ingram (16)
are a good example) has also played a part in the
persistence of a contentious literature on yeast
caryology. It is therefore of considerable interest
that a sharply defined ordinary-looking nucleus
has recently been described and photographed
in another member of the Saccharomycetaceae,
namely in Lipomyces lipofer (9), and that in
squashed preparations of budding cells of this
* To Professor Irene Manton., F. R. S.
yeast chromosome-like bodies, some of them in
pairs, have been seen arranged in what '~appeared
to be disturbed metaphase plates." It seemed
important to try to work out the course of mitosis
in a yeast with such favourable nuclei. A study of
the behaviour of the nucleus in growing and
dividing cells of Lipomyceswas therefore undertaken
and is described in the present paper. An account
of the nucleus of baker's yeast, which has been
studied at the same time, will follow.
The work of Ganesan and Roberts (9), which
forms the starting point of the present study, was
done on the nuclei of "giant cells" which regularly
arise in considerable numbers in cultures of
Lipomyces (29). The observations to be described
in the present paper have mainly been made on
the nuclei of cells of normal size. Their nuclei
behaved in the same way as the nuclei of giant
cells but were easier to study and photograph
because they were usually less obscured by stain
taken up by the cytoplasm.
MATERIALS
AND M E T H O D S
The Organism
A culture of Lipomyces designated CBS 944 was obtained from Dr. N. J. W. Kreger-van Rij at Delft,
who, with J. Lodder, has given the first definition
of this genus of the yeasts (19, 20). The organism was
879
g r o w n a n d m a i n t a i n e d on a m e d i u m c o m p o s e d of 2
per cent glucose, 0.5 per cent yeast extract (Difco),
a n d 1.5 per cent agar. G r o w t h was slow to start b u t
a b u n d a n t , a n d c o n t i n u e d for a long time. Stock
cultures on slants of this m e d i u m were kept at 4°C.
a n d were plated out a n d transferred to fresh slants
every two or three m o n t h s .
Microscopy of Living Cells
Cells from growing cultures were e x a m i n e d by phase
contrast microscopy in 16 per cent gelatine containing 1 per cent glucose a n d 1 per cent yeast extract.
This w a y of looking at living m i c r o o r g a n i s m s is derived from the work of Barer et al. (6) a n d in the
h a n d s of M a s o n a n d Powelson (21) has already provided m u c h useful information on the b e h a v i o u r of
the nuclei of living bacteria. T h e h i g h refractility of
the gelatin m e d i u m reduces the brightness of the
halos with w h i c h the phase contrast microscope surr o u n d s yeasts t h a t are e x a m i n e d in watery media. It
also increases the contrast between the nucleus a n d
t h e cytoplasm.
Fixation
Several fixatives were tried. T h e most instructive
preparations were obtained after fixation with the
fluid of Helly; with the s o d i u m sulfate of the original
formula omitted it is a solution of 5 per cent mercuric
chloride a n d 3 per cent potassium d i c h r o m a t e in water
to w h i c h 5 per cent formalin is a d d e d before use.
Helly fixation was chosen for two reasons: it has been
found very satisfactory by H e i m (11-15) in studies of
the nuclei of a wide r a n g e of fungi, a n d it was already
k n o w n to the present writer (from work to be p u b lished in a n o t h e r paper) t h a t Helly is a n excellent
preservative of the size, shape, a n d texture of the
nucleus of baker's yeast. O s m i u m tetroxide v a p o u r
a n d acetic acid-alcohol were also tried b u t were less
helpful because nuclei fixed by either of these reagents
were less easily resolved into c h r o m o s o m e s t h a n were
Helly-fixed nuclei.
T o m a k e preparations of Lipomyces, a loopful of
t h e slimy g r o w t h from a 2- to 5-day-old plate culture
was placed on a No. 1 coverslip. A second slip, its
corners t u r n e d t h r o u g h 45 degrees relative to t h e
first one, was placed on top. T h e drop of yeast was
allowed to spread a n d the two glass slips were t h e n
rapidly pulled a p a r t in opposite directions. T h e two
instantly drying films of yeast cells so obtained were
i m m e r s e d in t h e fixative. T e n m i n u t e s later t h e fixed
films were rinsed several times with 70 per cent alcohol a n d were t h e n transferred to small jars of the
m i x t u r e r e c o m m e n d e d by N e w c o m e r (27) for the
l o n g - t e r m preservation of tissues to be stained for
nuclei by the Feulgen technique. T h e s e jars were kept
in a domestic refrigerator.
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THE JOURNAL OF BIOPHYSICAL AND
It is a s h o r t c o m i n g of this procedure t h a t it allows
the cells to dry m o r e or less before they are fixed.
Preservation is indeed often poor in the thinly spread
" c o m e t ' s tail" of a film, w h e r e drying has been m o s t
rapid. Preservation is best in the thicker regions
a r o u n d the point of origin of the film. Brief exposure of
the yeasts to formalin v a p o u r before spreading t h e m
on coverslips has given e n c o u r a g i n g results, a n d the
possibilities of some such p r e t r e a t m e n t before proper
fixation deserve to be further explored.
Staining
A. HCl Giemsa Preparations:
T h e nuclei were studied
m a i n l y in G i e m s a preparations that h a d been treated
with N/I HC1 at 60°C. before staining. T h e usefulness of this kind of p r e p a r a t i o n in nuclear studies of
yeasts a n d filamentous fungi is n o w well established.
T o increase the contrast between c h r o m a t i n a n d
cytoplasm, fixed cells were usually first extracted for
11/~ hours with 1 per cent NaC1 at 60°C. as suggested
(with a slight difference in time, concentration, a n d
temperature) by G a n e s a n (8). T h e y were t h e n treated
for 10 m i n u t e s with N/1 H C I at 60°C., rinsed with
t a p water, a n d stained for several h o u r s in C o l u m b i a
staining jars (supplied by A. H. T h o m a s a n d Co.,
Philadelphia) containing 16 drops of G u r r ' s G i e m s a
R 6 6 (George T. G u r r Ltd., L o n d o n , S.W. 6, E n g l a n d )
dissolved in 10 to 12 ml. of G u r r ' s G i e m s a buffer
at p H 6.9. A few preparations were stained directly
with G i e m s a solution.
Stained films were differentiated by m o v i n g t h e m
a b o u t repeatedly for 10 to 12 seconds at a time in a
Petri dish with 40 ml. of distilled water to w h i c h one
wire loop, 2 m m . in diameter, of acetic acid h a d been
added. T h e acidulated water h a d a p H of 4.2. T h e
extraction of excess stain was controlled with a water
i m m e r s i o n lens. W h e n the nuclei stood o u t clearly
t h e p r e p a r a t i o n was m o u n t e d over a drop of buffer
containing 2 or 3 drops of G i e m s a per 10 ml. Excess
buffer was r e m o v e d by t o u c h i n g strips of bibulous
p a p e r to t h e edge of the coverslip. A sheet of such
p a p e r was t h e n placed firmly over the p r e p a r a t i o n
a n d while the first a n d second fingers of one h a n d
held the coverslip in place t h r o u g h t h e paper, h e a v y
pressure was several times b r o u g h t to bear on it with
t h e t h u m b of the other h a n d . T h e p r e p a r a t i o n was
t h e n sealed with wax, a n d either e x a m i n e d i m m e d i ately or first stored for several days in t h e refrigerator
at 4 - 6 ° C . Some films were sealed a n d e x a m i n e d
without h a v i n g been squashed.
B. Feulgen Test: Helly-fixed films were hydrolysed
for 10 minutes, rinsed with t a p water, a n d placed for
3~/~ h o u r s in Schiff r e a g e n t p r e p a r e d with D i a m a n t
Fuchsin ( C h r o m a G M B H , Stuttgart, G e r m a n y ) .
T h e y were t h e n rinsed quickly with 10 c h a n g e s of
SO2 water (tap water 90 ml., N/1 HC1 5 ml., 10 per
cent s o d i u m metabisulfite 5 ml.), followed by 20
BIOCHEMICAIJ CYTOLOGY • VOLUME
9, 1961
FIGURE 1
Schematic drawing illustrating the main steps in the proposed sequence of mitosis in Lipomyces. It
has been assumed that Lipomyces is normally haploid and has six chromosomes, but, as explained
in the text, this may not be right. In the absence of precise information it has been further assumed
that all the chromosomes are more or less of similar shape and size, though this again is unlikely to be
correct.
A. Resting nucleus with six single chromosomes and a nueleolus.
B. Beginning of prophase. The chromosomes have divided and arc long and thin.
C. Late prophase. The chromosomes have contracted. Arrows point at pairs of sister chromosomes.
The remaining ones may be regarded either as having not yet joined in pairs or as having been forced
apart by squashing.
D. Palisade or stack of paired sister chromosomes. Thc second and third pair from the top have
fused. The arrows indicate that the two gcnomes that are now aligned opposite each other have somehow to be separated and moved in opposlte directions. How this is achieved is not known. There is no
spindle.
E. Anaphase. The stack of separately visible pairs has fused into a seemingly homogeneous cord. At
its side is the gradually disappearing nucleolus.
F. Telophase. During anaphase the two genomes have moved to opposite ends of the cord, which now
divides transversely into daughter nuclei.
G. The new nuclei emerge from their dense telophase condition and can again be resolved into
chromosomes. A new nuclcolus has been formed.
minutes in running tap water, and were examined
mounted in water or in aceto carmine (23, 30).
Microscopy
Stained preparations were examined and photographed through a Zeiss microscope, 1935 model,
equipped with an achromatic condenser of N.A. 1.4,
apochromatic oil immersion lens X 90, N.A. 1.3, and
X 15 compensating eyepiece. Light from a tungsten
ribbon lamp was adjusted to give Koehler illumination, A Baird Associates interference filter was used
with maximum transmission at 5460 A, Photographs
were taken on Kodak Panatomic film at an initial
C. F. ROBINOW Mitosis in Yeast Lipomyces lipofer
881
magnification of 1800 and enlarged to ;< 3600 in
printing.
RESULTS
A. Observations on Living Cells (Figs. 2 and 3)
T h e cytoplasm of Lipomyces contained n u m e r o u s
watery vacuoles of different sizes, filamentous
mitochondria, shiny droplets of lipid, and small
dense granules whose n a t u r e has not been determined. T h e nucleus stood out clearly as a sphere
of low density containing a n excentric, dense,
r o u n d nucleolus. I n slide cultures prepared for
phase contrast microscopy cells of Lipomyces remained alive for m a n y hours in good condition,
b u t d u r i n g the time they were kept u n d e r observation their nuclei were not seen to divide.
B. Observations on Stained Preparations
T h e " c h r o m a t i c bodies" of Lipomyces, first seen
b y Ganesan a n d Roberts (9) a n d suspected by
them of being chromosomes, were found in the
nuclei of all cells of this yeast. T h e i r positive
Feulgen reaction c o m b i n e d with their affinity for
the Giemsa stain, their orderly behaviour, and
their occurrence in regular n u m b e r s leave no
d o u b t that they are indeed chromosomes. A
large n u m b e r of nuclei were studied until further
preparations no longer provided types of constellations of chromosomes t h a t h a d n o t been
seen before. T h e best-resolved photographs were
then arranged in w h a t the writer believes to represent a sequence of steps in the duplication of the
chromosomes and their distribution to d a u g h t e r
nuclei. These photographs have provided the
illustrations for this p a p e r and contain the m a i n
evidence for the account of mitosis that follows.
T h e principal features of the proposed sequence
are schematically illustrated in Fig. 1.
T h e cultures used in this work resemble those
which Roberts and Ganesan (29) have raised
Explanation of Figures 2 to 57
All photographs are of Lipomyces. All except Figs. 2 and 3 are from Giemsa preparations.
All except Figs. 2, 3, 6, and 56 are from squashed preparations.
The magnification everywhere is close to 3600 times.
FIGURES ~ AND 3
Living cells. In each cell can be seen a pale nucleus with dense nucleolus and vacuoles
both large and small. In Fig. 3 there is a shiny droplet of lipid next to the nucleus.
FIGURE 4
Young nuclei recently emerged from division in mother cell and fully grown bud.
Four to six chromosomes and a nucleolus can be made out in the upper nucleus.
FIGURE 5
Young nucleus in single cell as yet without bud. Four chromosomes are visible above
the nucleolus.
FIGURE 6
Budding cell with large nucleus in which four or five chromosomes can be seen. From
a preparation that had not been squashed. Comparable to Fig. 3 and, probably, Fig.
10, which is from a squashed preparation.
FIGURES 7 TO 14
Budding cells with nuclei in prophasc. Three single chromosomes and one or two
(newly formed?) pairs can be seen in the nucleus of Fig. 7. Fig. 8 illustrates a similar
stage. The chromosomes have duplicated themselves in Figs. 9 to 14. Six pairs of delicate
chromosomes surround the nucleolus in Fig. 9; four, perhaps five pairs in Fig. 10. Ten
to twelve tangled strands, some running parallel, can be counted in Figs. 11 and 13;
six pairs, already somewhat contracted, in Fig. 14-.
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C. F. RoI~INOW Mitosis in Yeast Lipomyces lipofer
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from single presumably haploid ascospores.' It is
therefore assumed that the present writer's cultures too are composed of haploid cells.
In the full grown bud or detached single cell
the nucleus is small and compact. It seems to
contain four to six slender chromosomes and a
nucleolus (Figs. 1, A ; 4, 5, and 49). Mitosis begins
with a long l~ropha~e d u r i n g ~ h i c h the chromosomes duplicate themselves in the intact and exp a n d i n g nucleus (Figs. 1, B; 6 to 14 a n d 57).
Towards the end of prophase the chromosomes
contract into minute shapes (Figs. 15 to 17) and
are later found in pairs (Figs. 18 to 28 and 56b;
Fig. 1C). An early stage of these changes is shown
in Fig. 24, a late one in Fig. 28. T h e origin of these
pairs is uncertain. Are they formed by independently moving isolated sister chromosomes? It is not
easy to see why sister chromosomes once formed
a n d cleaved a p a r t should later form a fresh association. It is easier to imagine that sister chromosomes are able to come together at this stage
because they have never completely lost contact
with each other. T h e wide scattering of m a n y
separate chromosomes in Figs. 15 to 24 seems to
argue against this view, but it must be r e m e m b e r e d
t h a t these are figures of nuclei that have been
m u c h flattened. Energetic squashing m a y have
forced chromosomes a p a r t that would have appeared joined in the intact nucleus (Figs. 56a,
56b); joined, perhaps, so it has been suggested
to the writer, by short lengths of non-staining
material. This attractive hypothesis would make
1 The formation of ascospores on yeast-water agar
(Roberts, 28) has been confirmed by the writer but
the behaviour of the nuclei during this process has not
yet been investigated.
the m e c h a n i s m of the events that followed easier
to u n d e r s t a n d than other schemes, b u t electron microscopy is required to either prove or disprove it.
Several factors combine to make counting of the
chromosomes in the photographs difficult: imperfect resolution, overlapping, insufficient d e p t h of
focus, tricks of perspective, and the fact that all
chromosomes in one nucleus apparently do not
undergo duplication, contraction, and the pulling
together into pairs at precisely the same time.
Where these shortcomings are least apparent, as in
Figs. 9, 15 to 17, and 19, ten to twelve chromosomes can be counted. T h e higher n u m b e r , six
chromosomes in the resting nucleus and six pairs
of sister chromosomes at late prophase, has been
tentatively accepted as n o r m a l for this strain of
Lipomyces. T h e various difficulties mentioned above
probably account for most instances where smaller
n u m b e r s are found. For details see legends to the
figures.
T h e pairs of chromosomes at first lie a r o u n d at
r a n d o m b u t later somehow arrange themselves
parallel to one another a n d form a stack or palisade. T h e cytoplasm a r o u n d the stacks is stained
as in the rest of the cell a n d no evidence has been
seen that a spindle a p p a r a t u s is involved in these
movements. This is not likely to be due to either
the fixative or the stain employed. Helly's solution is a good cytoplasmic fixative and spindles are
clearly visible in the HCI-Giemsa-stained p r e p a r a tions of meiotic nuclei of the perfect stage of
Helminthosporium recently described by K n o x Davies and Dickson (17). Half-way stages of the
process of a l i g n m e n t are illustrated in Figs. 29 to
35 a n d in Fig. 11 of G a n e s a n a n d Roberts (9),
finished stacks in Figs. 1, D, and 36 to 43. Counts
FrGURES 15 TO 17
Contraction of chromosomes in late prophase. Ten may be counted in Fig. 15, eleven
or twelve in Fig. 16, ten or twelve in Fig. 17.
FIGURES 18 TO ~8
Stages in the gradual reduction of the number of chromosomes through pairing. In
the figures not separately commented on, the chromosomes overlap so much that
reliable counts cannot be made. Fig. 18 may be interpreted in two ways. On the face
of it there are three large chromosomes, four smaller ones, and two very small ones,
making a total of nine. It is also possible to regard the three largest objects as pairs
of sister chromosomes with the remaining six still unpaired, giving a total of twelve.
Six complexes (pairs?), one including the nucleolus, are seen in Fig. 19. A constellation
suggestive of the beginning of a process of pairing is shown in Fig. 24. Five pairs and
two single dots can be counted in Fig. 27; five pairs also, if the two angular dots are
regarded as doublets, in the very similar Fig. 28.
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C. F. ROB][NOW Mitosis in Yeast Lipomyces lipofer
885
of the pairs of chromosomes in the stacks are likely
to be inaccurate for the reasons given above. T h e
n u m b e r s most c o m m o n l y found are four a n d five.
It is suggested that the stacks are composed of pairs
of sister chromosomes, and they are regarded as
m a r k i n g the metaphase of mitosis.
At anaphase (Figs. 1, E, a n d 44 to 46) the
chromosomes, surprisingly, come still closer together. They now cease to be separately visible
and a p p e a r to become fused into a single, solid
cord. A t telophase this divides into two dropshaped solidly c h r o m a t i n i c masses which move
away from each other b u t remain connected for a
while by a thin strand (Figs. 1, F; 47, 48, a n d 55).
A cell whose nucleus has reached telophase has
invariably grown a bud. Into this, one of the two
dense, angular sister nuclei now migrates. I t soon
unfolds into a small sphere complete with nucleolus a n d filamentous chromosomes (Figs. 1, G; 49,
and 4, which completes the circle).
T h e presence of a nuclear envdope can be inferred
from the rounded, compact a r r a n g e m e n t s of
chromosomes in early prophase, especially in
Fig. 4, and is obvious in electron micrographs of
sections of Lipomyces m a d e by Dr, R. G. E. M u r r a y
(personal communication). T h e nuclear envelope
remains inlact t h r o u g h most a n d perhaps all of
mitosis. This is obvious from the smoothly rounded
shape of the nuclei in all stages of division in
preparations t h a t have n o t been squashed. T h e r e
is a suggestion that the nuclear m e m b r a n e breaks
down before the stacks are formed. This is hinted
at in Fig. 1, C but awaits confirmation by electron
microscopy.
D u r i n g the greater part of mitosis the nucleolus,
as already noted by Ganesan and Roberts (9),
remains in contact with one or two of the chromosomes, as seen e.g. in Figs. 18, 31, 32, and 40. It
is later cast out into the cytoplasm and disappears
from view at the end of telophase.
Every culture of Lipomyces h a r b o u r s a considerable n u m b e r of cells that are very m u c h larger
than their fellows. As Roberts and Ganesan (29)
have already pointed out, the large or " g i a n t "
cells m a y have several nuclei of ordinary size or
one or two unusually large ones. A few giant
cells with large nuclei are shown in Figs. 50 to 55.
T h e chromosomes in the large nuclei are larger
(longer) b u t apparently not more numerous than
those in ordinary nuclei, a n d the two classes of
nuclei occupy the same volume at telophase
(Fig. 55).
C. Feulgen Reaction
T h e chromosomes are Feulgen-positive, most
strongly so at metaphase and telophase. T h e
FmI:RES ~29 TO 35
Prometaphase. Clusters of condensed and paired sister chromosomes, each pair having
the appearance of a short rodlet or dumbell, are beginning to align themselves for
inclusion in the palisade or stack of metaphase. The nucleolus, a soft-contoured round
body, less deeply stained than the chromosomes, can be seen among them or free in
the cytoplasm in all the figures. In Fig. 29 it has been drawn out into a crescent. Four
or five pairs can be counted in Figs. 31 to 34. Note the similarity between these four
figures. In all of them a single small body of chromatin is clinging to the nucleolus.
It is no longer found there fi'om anaphase onwards.
FIGURES
$6 TO 43
Metaphase. Four or five pairs of chromosomes are aligned more or less parallel to
one another in metaphase stacks. The alignment is most perfect in Figs. 36, 38, 39,
41, and 42.
FIGURES 44 TO 46
Anaphase. The several pairs of chromosomes of which a stack is composed now fuse
into a continuous cord.
FIGURES 47 TO 4 9
Telophase and beginning emergence of daughter nuclei.
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nucleolus, conspicuous in hydrolysed Giemsastained preparations, is entirely Feulgen-negative
and is invisible in Feulgen-aceto carmine slides.
D. Direct Staining
In preparations stained directly with Giemsa the
contrast between nuclei and cytoplasm was poor,
and only a few such slides were examined. Prophase
chromosomes appeared bright red and were more
delicate than in preparations stained after hydrolysis. Metaphase chromosomes were of a darker
purple or bluish red. Variation of the p H of the
stain might have produced better results, but the
course of mitosis, the main subject of the present
paper, was so much more easily followed in hydrolysed preparations that experiments with direct
staining were not continued.
DISCUSSION
The sequence of steps in the form of mitosis that
is claimed for Lipomyces is unusual and lacks the
support which precedent lends to schemes of
mitosis advanced for newly described species of
higher organisms. A summary of the logic behind
the proposed sequence might be useful at this
point. Neither the beginning nor the end of the
sequence is fixed arbitrarily. It is reasonable to
regard the chromatinic masses in Figs. 55, 47,
and 48 as illustrating the end of a process of
division which provided a nucleus for the bud
that has grown on the mother yeast. It is also
obvious that nuclei emerging from mitosis (Figs.
4, 5, and 49) are small and contain few chromosomes. Constellations of approximately twice that
number of chromosomes (Figs. 9, 10, 14, 16, and
17) have presumably arisen through the division
of the chromosomes of the original set and have
therefore been referred to as prophases. The
question now arises, by what means the duplicated
chromosomes are first sorted into two sister sets
and then separated. It is a far step from prophase
to the division stage of Figs. 46 to 48. An intermediate phase is required and is provided by the
palisades or stacks of Figs. 36 to 42. Long, narrow,
and densely chromatinic, the stacks merge
smoothly enough into the equally dense cords
and drop-like final division stages; their link with
prophase is less immediately obvious but consists
in their being composed of a small number of
chrornosomeqike subunits. Since that number is
approximately half that of the number of units in
the scattered prophase constellations, chromosomes must have either dropped out or joined with
others when the stacks were assembled. The fact
that the stacks (through the cords) give rise to
two nuclei forces us to accept the pulling together
of sister chromosomes as the event that precedes
the construction of the stacks. Evidence for such a
process is, I believe, to be found in Figs. 17 to 28.
The gradual transformation of a random cluster of
pairs of sister chromosomes into the tidy array of
the finished palisade is illustrated in Figs. 29 to 35.
The behaviour of the nucleolus corroborates
this story. Its position at a considerable distance
FIGURES 50 TO 5~
Giant cells. Prophase nuclei with long, thin entangled chromosomes.
FIOURE 53
Giant cell. Chromosomes contracted and scattered, approaching the stage of pairing.
Compare with Figs. 15 and 16.
F~GURE 54
Giant cell. Metaphase stack of four or five pairs of contracted chromosomes comparable in size to the stacks in Figs. 37 to 39 despite the great difference in the size
of the cells.
FIGVHE 55
Giant cell. Telophase. The nucleolus lies free in the cytoplasm between condensed
telophase nuclei. The size of the nuclei is the same as that of the telophase nuclei in
the much smaller cells of Figs. 47 and 48,
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C. F. RoBINOW Mitosis in Yeast Lipomyces lipofer
889
from the chromosomes in Figs, 29, 35, 36, and
41 to 43 makes it obvious t h a t these photographs
illustrate aggregates t h a t are formed after the
prophase constellations a n d do not represent
side views of them, as one m i g h t be tempted to
think by analogy with more conventional forms
of mitosis.
T h e close palisade of chromosomes at rectaphase a n d the even tighter cord of telophase may
perhaps be regarded as devices for the rapid and
accurate separation of two sets of chromosomes
without the help of a spindle apparatus. Because
they are already aligned opposite each other in
the stack, the two genomes can presumably be
gathered more swiftly t h a n if at metaphase their
members were still scattered at r a n d o m . T h e
relative scarcity of the cord stage in preparations
of Lipomyces indicates t h a t this phase is indeed
passed through very quickly. In this connection
it is interesting to note t h a t in Euglena, where a
spindle is also lacking b u t where the chromosomes
do n o t contract b u t r e m a i n relatively far apart
at metaphase, mitosis occupies several hours
(Leedale, 18). It is not k n o w n how the two
genomes which together form a stack slide past
each other, as they must, at anaphase-telophase
(Fig. 1, D). A possible a n d plausible device would
be first to transform the two genomes that confront
each other in the metaphase stack into two parallel
chains and then to develop some form of repulsion
between them. Confirmation of this idea must be
left to the electron microscope. 2
In a note published while this paper was being
written, Dowding and Weijer (7) state that in somatic
nuclei of Neurospora crassa the chromosomes at all
In achieving a proper distribution of its chromosomes without the aid of spindle fibres the nucleus
of Lipomyces does n o t differ from the somatic
nuclei of m a n y other fungi (1 5, 30, 32). W h a t
makes Lipomyces unusual is the clarity with which
its chromosomes are visible as separate bodies
through most of mitosis. T h e peculiar behaviour of
Lipornyces chromosomes which makes newly formed
sister sets move together r a t h e r than a p a r t at the
end of metaphase is probably also not restricted
to this yeast. T h e bouquets of contracted " c h r o m o somal filaments" which mark the halfway stage of
mitosis in Neurospora a n d Gelasinospora, according
to Bakerspigel (3, 4), are perhaps the result of similar manoeuvres. T h e example of Lipom),ces further
encourages a fresh look at other fungal nuclei
which late in their division assume the shape of a
densely c h r o m a t i n i c cord (or two of these parallel
and close together). Such cords which later divide
by elongation and constriction have been described
in Blastomyces a n d SchizophyUum (1, 5).
It would n o t be unreasonable to expect the
nuclei of baker's yeast to resemble those of Lipomyces. In reality they are different. T h e nuclei of
b u d d i n g yeast cells, fixed a n d stained by the
methods used in the present work, have at all
times a fine-grained structure and, at the level
accessible to the light microscope, do n o t divide
by the form of mitosis found in Lipomyces. In corntimes hang together in a chain which splits lengthwise
in the course of mitosis. This view is at variance with
the results of two other recent studies of the nuclei of
this fungus by Bakerspigel (3) and Somerset al. (31),
who also differ among themselves on many points of
interpretation.
FIGURES 56a, 56b, AND 57
These figures are intended to give an impression of the general character of Lipomyces
preparations that cannot be obtained from the preceding small figures of isolated
cells. They also serve to illustrate the difference between intact nuclei and
squashed ones.
F1GI~RES 56a AND 56b
Two different levels of focus of the same group of cells of Lipomyces in a preparation
that had not been flattened. In the centre of both pictures is a cell with a nucleus in
prophase. Arrows point to cells with nuclei at that stage of mitosis where the chromosomes shorten and pairs begin to emerge. These nuclei are comparable to the squashed
ones in Figs. 25 to 28.
FIGURE 57
Cells with nuclei in prophase. This preparation had been squashed.
890
T h E JOURNAL OF BIOPIfYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME ,0, 1961
C. F. ROBINOW Mitosis in Yeast Lipomyces lipofer
891
m o n with M c C l a r y et al. (22) I have seen countable chromosomes in baker's yeast only at meiosis.
This work will be described in a separate paper.
This work was supported by a grant from the National Research Council of Canada.
Received for publication, January 22, 1961.
The author is indebted to Mrs. Elizabeth Berry for
several useful suggestions.
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THE JOURNALOF BIOPHYSICALANDBIOCHEMICALCYTOLOGY - VOLUME9, 1961