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Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum
DEVELOPMENTAL
Nuclear
BIOLOGY
34, 228245
(1973)
DNA Content Myxomycete
and Chromosome Numbers Physarum polycephalum
in the
JOYCE MOHBERG, KARLEE L. BABCOCK, FINN B. HAUGLI, AND H. P. RUSCH McArdle
Laboratory,
Medical
School, University Accepted April
of Wisconsin, Madison,
Wisconsin 53706
20, 1973
An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum. It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein. Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20-25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35-40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein : DNA and RNA: DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker. A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined nlasmodial growth medium, containing 2% agar. Plasmodia are visible in 2-4 days. INTRODUCTION
The myxomycete, Physarum polycephaZum, would seem ideally suitable for biochemical studies of mitosis and meiosis and of haploid and diploid nuclei, since plasmodial mitosis and meiosis are both synchronous (Howard, 1931; Guttes et al., 1961; Aldrich, 1967; Arescaldino, 1971) and all stages of the life cycle can now be cultivated axenically (Daniel and Baldwin, 1964; Goodman, 1972). However, such investigations are meaningful only if the particular culture line under study actually undergoes meiosis, and it is clear that the mere fact that spores are formed does not prove that meiosis has occurred, since sporulation can occur without meiosis (von Stosch et al., 1964), meiosis I can occur without spore cleavage (Aldrich and Carroll, 1971) or meiosis I can be arrested until shortly before spore germination (Cathcart and Aldrich, 1972). In order to find a means of determining whether our cultures were completing meiosis, we did DNA analyses throughout the life cycle in sev-
era1 of our established sublines and found that those giving viable spores had twice the DNA content in the plasmodial nucleus as in the spore, whereas a culture giving nonviable spores had the same DNA content in the plasmodium as in the spores, and apparently did not undergo meiosis (Mohberg and Rusch, 1971). We now have extended the study to a series of sublines which have been growing in the laboratory for shorter periods of time, either because they had been kept as sclerotia since shortly after purification from field collections, or because they had arisen from recent matings of amoebae. This time we have done chromosome counts in plasmodia and germinating spores in addition to DNA analyses at different stages of the life cycle, and the data presented in this paper are consistent with the heterothallic strains being haploid in the amoeba and diploid in the plasmodium. Among the new matings of amoebae used in this study, several were found which had abnormally large nuclei. Al-
228 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
MOHBERC
et ~1. Chromosome
though most of them did not sporulate, these cultures were still of value because they provided an opportunity for studying nuclear: cytoplasmic relationships without subjecting the organism to such trauma as amputation (Prescott, 1956b) or exposure to visible (Prescott, 1956a) or ultraviolet light (Sachsenmaier et al., 1970). This paper gives data on chromosome numbers, on the relation of nuclear DNA and protein to nuclear volume and surface area, and on plasmodial protein : DNA and RNA : DNA ratios in normal and polyploid cultures. MATERIALS
AND
METHODS
Culture strains and methods. P. polycephalum amoebae clones RSD 2 through 9
DNA
CONTENT
OF PLASMODIAL
Numbers
were obtained by Rodger Steeper of this laboratory from spores of a plasmodium (RSI) grown from the Wis 1 isolate of Dr. M. Backus of the Botany Department of this university. Amoebae a, i, B173, B174 (Dee, 1962, 1966) and the homothallic (Wheals, 1970) strains C 5-1 and C 50 were the gift of Dr. J. Dee of the Genetics Department of the University of Leicester. Amoebae were grown on Escherichia coli lawns on agar or on Millipore membrane (Haugli, 1971) and were mated to give the plasmodia listed in Table 1. RSD amoeba clones were shown by Haugli (1971) to be of mating types 1 and 2 (Dee, 1962), and matings are referred to subsequently by number only, the mating type 1 parent
TABLE I NUCLEI AND OF SPORES OF DIFFERENT
Plasmodium pg per nucleus
7x 9x 5x M,ad axi ix B173 B173 x RSD8 B173 x B174 i x B174
SUBLINES
OF Physarum
Polycephalum
DNA Content
Subline
M,cVII M,cVII (Be) M,cIV c 5-1 c 50 RSP RSD4 x RSD 4x 5x 9x 4x 5x
229
in Physurum
6 8 6 8 3 3; 1st mating 2nd mating 3 3 2
1.23 1.35 1.15 1.07 0.88 0.97 1.21 1.34 1.13 1.04 1.92 1.88 1.87 1.87 1.84 3.64 0.91 0.94 1.18 1.16 1.77 1.94
* 0.08(4)” * 0.02(2) * 0.09(5)” f 0.03(6) + 0.03(5) * 0.03(4) * 0.14(4) * 0.02(3) f 0.08(7) * 0.07(6) * O.ll(5) + 0.05(5) * 0.17(5) * O.lO(5) zt 0.06(6) zt 0.19(6) * 0.09(4) * 0.05(7) f 0.01(2) f 0.04(2) * 0.13(5) * O.OO(2)
Spore pg per spore 0.62 + 0.03(5) 0.53 1.17 i 0.06(4) Nonsporulater 0.92 * O.OO(2) 0.65 * 0.06(4) Nonsporulater 1.09 * 0.10(2) Nonsporulater 0.60 i 0.04(5) Nonsporulater Nonsporulater Nonsporulater Nonsporulater Not analyzed Not analyzed 0.64 + 0.06(4) 0.74 * 0.01(2) Not analyzed Not analyzed Not analyzed
a Plasmodial nuclei were analyzed at 4-6 hr after mitosis and spores at l-2 weeks after cleavage, Nuclei and spores were washed and counted as described in Materials and Methods. DNA content is given in picograms per nucleus or spore + standard error; the number of preparations analyzed is given in parentheses, h From Mohberg and Rusch (1971). c Parent of RSD amoeba clones. d Parent of a and i amoeba clones.
230
DEVELOPMENTAL BIOLOGY
being given first. Other plasmodial sublines used were CL, a gift of J. Dee; Wis 2, a second strain collected by Dr. Backus; several M,‘s from an earlier purification of the Wis 1 isolate (Daniel and Baldwin, 1964); M,cVII(IIe), obtained by passing M,cVII through the life cycle several times (Haugli, 1971); 9 x 8 “h”, a possible homothallic obtained by Dr. D. N. Jacobson of this laboratory from spores of nitrosoguanidine-treated 9 x 8 plasmodium; and 9 x 8 “h” fz, a polyploid obtained by passing 9 x 8 “h” through the life cycle twice. Physarum flavicomum plasmodia were provided by Dr. W. M. LeStourgeon of this laboratory. Stock cultures of all plasmodia were carried as microplasmodia in submersed culture in semidefined medium (Daniel and Baldwin, 1964), and were stored as spherules. The M, sublines, RSI, a x i and the homothallic sublines were induced to spherulate by transferring microplasmodia to salts-citrate medium (Daniel and baldwin, 1964), but other sublines lysed in that solution and were therefore allowed to spherulate in exhausted nutrient medium as follows: Five milliliters of medium in 20 x 150-mm tubes were inoculated with microplasmodia and incubated for 10 days to 2 weeks on a gyratory shaker (New Brunswick, Model V with l-inch stroke) at 200 rpm at 23”-26°C. Tubes were then either stored’ at 4” or spherules were pelleted, washed with water, dried on filter paper, and stored in a desiccator at 4°C. Synchronous plasmodial cultures were grown in petri dishes on filters supported by stainless steel screen as described earlier (Mohberg and Rusch, 1969) except that Millipore membrane was used only for M, sublines and Schleicher and Schuell 576 filter paper was used for growing other sublines and for sporulating all sublines. Heterokaryons were made by inoculating dishes with equal amounts of microplasmodia of two sublines, as described by Haugli et al. (1972). Under these conditions all sublines fused initially but non-
VOLUME 34, 1973
compatible combinations-5 x 2 and M,cVII(IIe), for example-separated later, and sublines were therefore judged compatible only if they still gave a single disk-shaped plasmodium at 36 hr after inoculation. [See Haugli (1971) and Dee (1973), for plasmodial fusion characteristics of some M,c derivatives.] Sporulation was induced by illuminating plasmodia which had been starved on niacin-carbonate medium, either according to Daniel and Baldwin (1964) or to Haugli et al. (1972), or which had been starved on exhausted nutrient medium, made initially with 0.5 instead of 1% (w/v) glucose. The latter medium was always used with subline a x i because it sclerotized on the salts medium and was used with M, sublines whenever rapid spore germination was needed. Plasmodia were put into the illuminator (Daniel and Rusch, 1962) 4-7 days after inoculation and were illuminated for 4 hr each day until they sporulated. Spores were germinated by scraping sporangia (l-3 weeks after cleavage) into 40 ml of distilled water and crushing in a Potter-Elvehjem homogenizer with Teflon pestle. (Washing was done aseptically at room temperature.) The spores (25-75 million) were centrifuged at 1000 g for 5 min and washed three more times with water. They were then suspended in 5 ml of water and put into a petri dish. Germination in M,cVII(IIe) and 9 x 8 spores began within half an hour, with the highest proportion of metaphase figures occurring at 1 to 1.5 hr. CL and C 50 were slower with germination beginning in about 3 hr. To complete the life cycle, spores were left in water for 6 hr, by which time germination was finished, and l-ml aliquots were put into petri dishes containing 20 ml of one-tenth strength semidefined plasmodial growth medium (Daniel and Baldwin, 1964) in 2% agar. Spores were spread with a glass rod and the dishes were incubated at 26”. Alternatively, spores were put on Millipore membrane supported by a stainless steel grid
MOHBERC
et al. Chromosome
over diluted medium. With either system plasmodia were visible in 2-4 days. Chromosome counts. The mitotic stage of growing and sporulating plasmodia was determined by examining smears (Sachsenmaier and Rusch, 1964; McCormick and Nardone, 1970) with the phase contrast microscope. When nuclei of growing plasmodia entered prometaphase, cultures were cut in half and one half was immediately dipped in ice-water and homogenized (see below). The other half was harvested 2 min later. Since chromosomes spread well only during the middle 2-3 min of metaphase, 4-6 nuclear preparations were made to ensure that at least one would give usable spreads. Sporulating plasmodia were cut into fourths when they entered prophase and the segments were harvested at 15min intervals, since metaphase seemed to last longer than in the growing plasmodium and mitosis was not as tightly synchronous. Metaphase nuclei were isolated from growing plasmodia by slow homogenization in 40 ml of nuclear isolation medium (for details of equipment and medium composition, see Mohberg and Rusch, 1971). The suspension was passed through milk filter, divided equally between two conical centrifuge tubes and underlaid with 10 ml of 1 M sucrose solution. Tubes were centrifuged at 50 g for 15 min and the pellet was removed with a wide-tipped pipette. Nuclei were then pelleted by centrifuging at 1000 g for 15 min. After they had been washed once in 40 ml of nuclear isolation medium, they were suspended in 0.2 ml of the same solution by vortexing and were kept in ice while chromosome spreads were being made. Sporangia were washed with 0.25 M sucrose, containing 0.01 M ethylene diaminetetraacetate (pH 7.5) and were homogenized in 40 ml of nuclear isolation medium with 4 or 5 strokes in a Potter-Elvehjem homogenizer, followed by 2 min of slow stirring with a Waring Blendor. Nuclei were isolated from the homogenate as
Numbers
in Physarum
231
described for growing plasmodia except that they had to be passed through 1 M sucrose at least twice before they were sufficiently free of cytoplasmic granules (lipid and/or polyphosphate?) so that chromosomes would spread properly. Chromosome spreads were made by a modification of the method of LaCour (1941). A drop of nuclear suspension was mixed on a slide with 2 drops of lactic acid (2 parts of 88% lactic acid mixed with 1 part of water), and was heated over a small flame. (We are indebted to Dr. John Ellison, formerly of the Anatomy Department of this university, for this suggestion.) The slide was allowed to cool for several minutes, and 2 drops of 2% orcein in 75% acetic acid were added. The mixture was warmed again and covered with a cover slip. Five minutes later the preparation was flattened between several layers of paper towel. Observations and photographs were made with a phase contrast microscope within 12-24 hr. (Orcein stain was made by dissolving 2 gm of Baker synthetic orcein in 50 ml of boiling glacial acetic acid, and diluting the stock solution with 45% acetic acid immediately before use. The mixture was then centrifuged and filtered.) Analytical methods. Nuclei and spores were suspended in 0.25 M sucrose, containing 0.01 M CaCl,, and were counted in a hemacytometer or measured with a micrometer. An equal volume of 10% (w/v) trichloroacetic acid in acetone was then added to the nuclear suspension, and it was centrifuged at 75,000 g. The pellet was washed twice with 0.25 M perchloric acid at 0” and extracted twice with 0.5 M perchloric acid at 70”. DNA and protein were analyzed by the methods of Burton (1956) and Lowry et al. (1951) with the modifications described previously (Mohberg and Rusch, 1969). RESULTS
DNA Content of Plasmodial
Nuclei
Most of the sublines used in this study
232
DEVELOPMENTAL BIOLOGY
(Table 1) were “new”; that is, they had arisen from recent mating of amoebae or, as in the case of RSI and M ,cVIII, had been kept as spherules since shortly after purification from the Backus Wis 1 sclerotium. MscIV was included as an “old” culture because during 4 years of cultivation in the laboratory it had lost the ability to complete meiosis but not to sporulate (Mohberg and Rusch, 1971). M,a was included because we had two stocks, one (“new” M,a) which had been grown in the laboratory for a short time before it was stored as spherules (in 1962) and another (“old” M,a) which had been grown for several years before storage. We do not know which of these M,a’s is equivalent to Ross’s 51D (Ross, 1966). Nuclear DNA content (Table 1) was roughly 1 pg in the M, sublines, in most of the RSD and Dee amoeba crosses and in the homothallic strains C 5-1 and C 50. Diameter in these nuclei was between 3.0 and 3.5 pm. All matings of RSD 3 and of B174 had a DNA content of about 1.8 pg and a diameter of 4-4.5 pm. The one mating of RSD 2 which was analyzed had a diameter of about 6 pm and a DNA content of 3.6 pg. These sublines with higher nuclear DNA content grew somewhat slower than did M,c, but mitoses were synchronous and similar to those of normal cultures
except that nucleolar reconstruction was slower. The larger nuclei had larger nucleoli, and late interphase 9 x 3 and 5 x 2 had nucleolar diameters of approximately 2.5 and 3 pm, respectively, as compared to 1.8 pm in M,c. When the data of Table 1 were arranged as in Table 2, it appeared that the DNA content of amoebae RSD 4 through 9, provided that they mated 1 to 1, must have been about 0.6 pg, which agrees with the value of 0.55 pg found in nuclei of log phase RSD 4 and RSD 5 (Mohberg and Rusch, 1971). Amoebae RSD 3, however, appeared to contain 1.2 pg of DNA and RSD 2, 3 pg. Similarly, analyses of crosses of the Dee amoebae suggest that a, i, and B173 all contained 0.6 pg of DNA and B174 contained 1.2 pg like RSD 3. We have not done DNA analyses of any amoeba clones except 4 and 5 because, as reported before (Mohberg and Rusch, 1971), agar-grown cells cannot be used because the agar interferes with the diphenylamine reaction; and some clones, such as RSD 2 and 3, grew erratically on Millipore membrane. However, RSD 2 seemed definitely polyploid, judging from its large cell size and nuclear diameter, which was 3.8 pm, as compared to less than 3 in RSD 5. Sachsenmaier et al. (1970) have ob-
TABLE PREDICTED DNA Amoeba sublines mt, RSD 4
2
CONTENT OF NUCLEI OF RSD AMOEBAE” 8
DNA per amoeba nucleus ( pg)
0.6" 0.6 0.7 0.6
Amoeba sublines; mt2” RSD2
3
DNA per plasmodial -
6
nucleus (pg)
-
1.9 1.9 1.9 1.8
1.2 1.1 -
1.3 1.1
3.0
1.2
0.6
0.6
5
3.6
7 9 DNA per amoeba nuc-
VOLUME 34, 1973
leus (ps) a DNA content of plasmodia derived from RSD amoebae has been tabulated according to parent amoeba of each plasmodial subline, with mating type 2 parents across the top of the table and mating type 1 down the left side. The predicted DNA content of the mt, amoebae is in the right-hand column, and of the mt, amoebae across the bottom of the table. DThese values were obtained by analysis of isolated RSD 4 and 5 amoeba nuclei (Mohberg and Rusch, 1971).
MOHBERC et al. Chromosome
tained data from studies of UV-irradiated plasmodia which suggested that P. polycephalum resembles Amoeba proteus in having a critical cytoplasm : nucleus (protein : DNA) ratio (Prescott, 1956a, b) which must be reached before nuclear division can occur. It was therefore of interest to determine the protein : DNA and RNA: DNA ratios for whole microplasmodia of sublines with different nuclear DNA contents. Both protein : DNA and RNA : DNA ratios were roughly equal for all sublines tested (Table 3), indicating that those cultures with larger nuclei had proportionately fewer of them. Pigment : DNA ratios differed markedly among the various crosses and ranged from lemon yellow in 5 x 2 and 9 x 8 “h” f, to orange in 9 x 3 and 9 x 8. This was useful when microplasmodia of different sublines were mixed to make heterokaryons (see Materials and Methods) because noncompatible pairs, such as 5 x 2 and M,cVIII, could be distinguished both by the colors of the TABLE PROTEIN, RNA,
Numbers
233
in Physarum
plasmodia and by the size of the nuclei. In the myxamoebae of Dictyostelium discoideum (Sussman and Sussman, 1962) and in the amoeba Naegleria (Fulton, 1970), nuclear volume is proportional to DNA content. On the other hand, the rate of DNA synthesis in plant and animal cells appears to be related to nuclear surface area (Alfert and Das, 1969). We therefore plotted the surface area and volume of nuclei of different sublines against both DNA and protein content. Figure 1 shows that both DNA and protein content gave essentially straight lines when plotted against both volume and surface area. However, only DNA vs surface area passed through the origin. The other three curves all intercepted the y axis. DNA Content of Spores
Most of the sublines with a nuclear DNA content of about 1 pg sporulated, and all but 4 x 8, C 50 and M,cIV gave spores which contained 0.5 to 0.6 pg of DNA when 3
AND PIGMENT CONTENT OF MICROPLASMODIA OF SUBLINES WITH DIFFERING NUCLEAR DNA CONTENT
Subline
M,cIV axi 9x8 Mean B173 x B174 i x B174 4x3 9x3 9 x 8 “b” f, Mean 5 x 2; 1 yr in culture 5x2
DNA per nucleus (PP)
Composition Protein/DNA ~ccdrg)
of microplasmodia” RNA/DNA (KdPP)
1.2 1.0 1.1
85.9 85.3 81.8
13.3 13.0 10.8
1.1
84.3
12.4
1.8 1.9 1.9 1.8 2.0
70.5 70.5 91.3 78.4 84.7
10.8 Not done 13.4 12.0 9.8
1.9
79.1
11.5
2.7 3.6
92.1 75.5
13.7 11.7
PigmentiDNAD 0.24 0.55 0.62
0.46 0.54 0.67 0.81 0.30
0.26 0.29
a Microplasmodia in early to mid log phase growth were pelleted, extracted with TCA-acetone to remove pigment, and were washed with cold perchloric acid and extracted with hot PCA as described earlier (Mohberg and Rusch, 1969). Each value represents the mean of analyses of two to four samples. b Pigment is expressed here as optical density at 415 nm of 1 ml of TCA-acetone extract in a 13-mm light path (Daniel and Baldwin, 1964).
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DEVELOPMENTAL BIOLOGY
VOLUME 34, 1973
B174 were ever observed to fruit. DNA analyses were not done on spores of either subline but chromosome counts were made in B173 x B174 during sporulation. Results are in the following section. Chromosome Plasmodia
0x1 -t-
0
I
I
40 80 VOLUME;
I
120 p3
I
0
I
I
40 80 120 SURFACE AREA; ~~
FIG. 1. Relation of nuclear volume and surface area to DNA and protein content. Nuclear diameters of plasmodial nuclei at 3 hr after mitosis were plotted vs frequency, as in Figs. 6 and 8. Nuclear volumes and surface areas were calculated from the areas under the diameter curves and were plotted vs DNA and protein content in picograms per nucleus. Curves were fitted by the method of least squares (Young, 1962). Points for normal-sized nuclei are labeled in the right-hand panels and points for 5 x 2 and the matings of amoeba RSD 3 with RSD 4, 5, 7, and 9 are labeled in the left-hand panels.
mature. (Germination in 4 x 8 and C 50 was 10-15s and in M,cIV, less than 0.1s.) Table 4 shows that the DNA content of spores remained at about 1 pg per spore for the first 24 hr after cleavage, and then slowly decreased during the next several days to a final value of about 0.6 pg, where other experiments showed that it remained for at least the next 3 weeks. Spores were capable of germinating by 4 days after cleavage, and swarm cells began to emerge within 30 min of plating. Germination was complete in about 3 hr. Amoebae divided before, during and after spore germination so that by 5 hr after plating there were two amoebae for each spore that had hatched. At this time the myxamoebae from 9 x 8, the only subline yet analyzed, contained 0.55 pg of DNA per nucleus. Of the sublines with a DNA content of 1.8 pg and above, only 5 x 2 and B 173 x
Counts
in
Growing
Ross (1966) and Kerr (1968) both reported having considerable difficulty in making chromosome spreads with whole slime mold plasmodium. Our own earlier efforts showed that the cytoplasm not only kept the nuclei from flattening adequately, but also seemed to interfere with staining of the chromosomes. We therefore tried using isolated metaphase nuclei as starting material because mitosis is intranuclear (Figs. 2A and 2B), and it should therefore be possible to get rid of cytoplasm without losing chromosomes. This proved successful, provided that nuclei were heated in lactic acid to spread the chromosomes before stain was added. Germinating spores and growing amoebae were not fracTABLE
4
EFFECT OF AGE OF SPORESON THEIR DNA CONTENTS Age of spores (hr post-cleavage)*
Sublines” 9 x 8’
M,cVII(IIe)’
DNA content (pg per spore)
12 to
1 4 24 48 72 96 144 15 days
1.11 Not done 0.86 0.80 Not done 0.79 0.68 0.60 + 0.04(5)
Not done 0.94 0.91 Not done 0.63 Not done 0.67 0.53
o Spores of 9 x 8 and of M,cVII(IIe) were produced in niacin-salts medium and harvested at the times indicated. Spores were washed, counted, and analyzed as described in Materials and Methods. b Mitosis occurred at 13 hr after the end of illumination, spore cleavage 0.5 to 1 hr after mitosis, and melanization of spores at about 1 hr after cleavage. c DNA content of plasmodial nuclei (Table 1) was 1.04 * 0.07(6) in 9 x 8 and 1.35 + 0.02(Z) in M,cVII(IIe).
MOHBERG
et al. Chromosome
Numbers
in Physarum
235
FIG. 2. Photomicrographs of starting material for chromosome spreads. (A) Isolated metaphase nuclei of growing plasmodia of M,cIV (left) and of 5 x 2 (center) and of a sporulating plasmodium of M,cVIII (right). Nuclei were isolated as described earlier (Mohberg and Rusch, 1971) and were stained with acetic-orcein and photographed with phase contrast. (B) Electron micrograph of isolated metaphase nuclei of a growing M,cIV plasmodium. Isolated nuclei were fixed with glutaraldehyde and stained with osmic acid and lead, according to Reynolds (1968). (C) Germinating spores of M,cVII(IIe), about 2 hr after they were plated as described in Materials and Methods. Phase contrast: unstained. (D) Higher magnification of metaphase in a germinating spore. Acetic-orcein stained; phase contrast. A and D, x2300; B, ~22,000; C, x500.
236
DEVELOPMENTAL BIOLOGY
tionated but were spread by heating the whole cells in lactic acid, since the nuclear membrane disappears during mitosis in the amoeba (Kerr, 1967; Aldrich, 1969). Chromosome spreads of nuclei from different stages of the life cycle are shown in Figs. 3 and 7. In the growing plasmodium most chromosomes were very similar in size and shape, but there were some distinctively different chromosomes, such as the very long ones at 4 to 5 o’clock in Fig. 3A. Banding along the chromosomes was evident in many spreads, and it was expected that certain of these bands might be stained with quinacrine (Ellison and Barr, 1971), but Dr. Ellison (personal communication) was unable to find any quinacrine-positive regions in our preparations. Somatic association (Feldman et al., 1966) could be seen in spreads of both growing (Figs. 3A and 3B) and sporulating plasmodia. In late metaphase, chromosomes sometimes could be spread sufficiently thin so that individual chromatids could be seen (Figs. 3C and 7G). It is obvious from Fig. 3 that the number of chromosomes per spread differed markedly, even among sublines a x i, M,a, and M,c, all of which had similar nuclear DNA contents. Histograms of chromosome number (Figs. 4 and 8) revealed that chromosome number varied as much within a subline as among sublines. In “old” M,a and M,cIV, both of which had been growing in the laboratory for a long time, most contained 50-55 chromosomes, nuclei similar to Ross’s 51D (Ross, 1966). In RSI (Figs. 4 and 5B) and “new” M,a (not shown) about 25%, and in M,cVIII and M,cVII(IIe) (Figs. 7A and 8) almost 50%, of the nuclei had 75 or more chromosomes. On the other hand, in a x i 25% of the nuclei had 25-30 chromosomes (Figs. 3A and 4), and in the homothallic cultures essentially all nuclei had 35-40 chromosomes (Figs. 4 and 9). In P. flauicomum (not shown) the major peak was between 80 and 90 chromosomes, which is somewhat higher than the 70 found by Ross (1966),
VOLUME 34, 1973
and there were smaller peaks at about 50, 70, and 100. Among the sublines with a higher nuclear DNA content, 9 x 8 “h” f, had two ploidy levels, one with 150 (Fig. 5D) and one with 175 chromosomes (Fig. 6). In 4 x 3 there were peaks at 75, 100, and 150 chromosomes and in 9 x 3 at 75 and 100 (Fig. 5C). In 5 x 2 there were between 150 and 240 chromosomes in the majority of the nuclei. This probably is owing both to the presence of several ploidy levels and the difficulty of flattening very large nuclei sufficiently for all chromosomes to be seen without at the same time stretching and breaking others. Both 9 x 3 and 5 x 2 contained a few nuclei with 50 chromosomes, and these presumably survive better under adverse conditions than do the larger nuclei, thus resulting in the occasional drop in nuclear size that was seen with polyploid cultures (see later section). Chromosome
Counts in Sporangia
Although both M,cVIII and RSI fruited readily, chromosome counts on the precleavage nuclear division were done with sublines M,cVII(IIe), 9 x 8 and 9 x 8 “h,” since the spores of these cultures gave 50-60% germination, as compared to 5-10% in M,cVIII and RSI, and it could therefore be assumed that at least half of the nuclei which divided before spore cleavage went on to complete meiosis. Since a x i and the Colonia strains have been used frequently in genetic studies (Wheals, 1971; Dee, 1973), these cultures also were included, although the spore germination was too slow to permit counting chromosomes in hatching amoebae. In both the heterothallic (Fig. 8) and the homothallic (Fig. 9) strains the same ploidy levels were seen in the sporangium as in the growing plasmodium. Germinating spores of “new” M 3a, M,cVII(IIe), 9 x 8, and 9 x 8 “h” had 20-25 chromosomes, indicating that reduction had occurred at some time between spore cleavage and spore germination.
MOHBERG et al. Chromosome
Numbers
in Physarum
237
FIG. 3. Photomicrographs of representative chromosome spreads. Metaphase nuclei were from growing plasmodia for all spreads except H and I. (A) a x i, showing individual chromosomes. (B) 4 x 3, showing somatic association. (C) 9 x 8 “h” f,, showing splitting chromosomes. (D) M,a (Ross’s 5lD), 47 chromosomes. (E) P. fkxuicomum, 86 chromosomes. (F) C 50, 40 chromosomes. (G) M,cVIII, 80 chromosomes. (H) M,cVII(IIe) germinating spore, 22 chromosomes. (I) Growing amoeba from spores of the 5 x 2-9 x 8 heterokaryon, 30 chromosomes. All spreads are at x 2300.
238
DEVELOPMENTAL BIOLOGY
VOLUME 34, 1973
Factors Affecting
FIG. 4. Chromosome numbers in representative plasmodial sublines. Chromosome spreads were made of growing plasmodia of the sublines indicated on the graph. Chromosomes were counted for a number of spreads for each subline, and counts were grouped into intervals of 5. A histogram was then made with frequency, as percent of total spreads counted, vs chromosome number. The number of spreads counted were70ofM,cVIIandC5-1,50ofa x i,40ofM+and RSI, and 25 of M,cIV.
As mentioned above, the only polyploid subline from Table 1 which fruited readily was B173 x B174, which had about 75 chromosomes during growth and sporulation (Fig. 10). Spores germinated too slowly for counts to be made in hatching amoebae. Since 5 x 2 could no longer be induced to sporulate after the method for counting chromosomes had been worked out, we resorted to making a heterokaryon with 9 x 8, which fruited very readily. In the precleavage mitosis (Fig. 6, dashed lines in bottom panel) the majority of the nuclei had 50 and 75 chromosomes like the 9 x 8 parent, and only a small fraction had the ploidy of the 5 x 2 parent. Moreover, the nuclear diameter in the plasmodium which arose from the heterokaryotic spores was about 3.5 pm, the same as in 9 x 8. Similar results were obtained when a 9 x 3-9 x 8 heterokaryon fruited. Recently we have obtained a derivative of M,cVII(IIe) which has 100-110 chromosomes and sporulates readily, giving polyploid amoebae. This culture was grown from spores which were heated to 37” for 10 min during the washing procedure (see Materials and Methods.)
Ploidy
On several occasions nuclear size of cultures dropped spontaneously. One example was a 5 x 2 culture which during a year of cultivation dropped from 3.6 to 2.7 pg of DNA per nucleus. Other examples were 5 x 2, 7 x 3, and B173 x 174 cultures which had normal-sized nuclei after they were freed of an unidentified contaminant by migrating across water agar. Results in the preceding section suggested that sporulation might select against polyploid nuclei. When B173 x 174 was passed through spores, the resulting plasmodium had a higher percentage of nuclei with 50 chromosomes, but when M,cVII(IIe) was handled similarly, the percentage of nuclei with 75 chromosomes did not decrease., Recloning amoebae before mating also was ineffective in reducing the fraction of polyploid nuclei. As mentioned above, an increase in ploidy was obtained when spores were heated before germination. DISCUSSION
The main problem in making chromosome spreads of isolated metaphase nuclei of the synchronous plasmodium of P. polycephalum is that metaphase lasts only about 7 min (Mohberg and Rusch, 1971), and it is therefore difficult to judge harvesting time so that nuclei are arrested in mid-metaphase when chromosomes are condensed and before they split into chromatids. However, once a good preparation has been made, it gives dozens of usable spreads on a single slide. Myxamoebae are much more difficult to work with because cultures do not have natural synchrony and metaphase is of about the same duration as in the plasmodium, so that even very rapidly proliferating cultures have only a small percentage of nuclei in the portion of metaphase giving good spreads. It appears that before reliable data can be obtained it will first be necessary to devise a technique either to synchronize amoebae or to block
MOHBERC et al. Chromosome
Numbers
in Physarum
239
FIG. 5. Chromosome spreads of polyploid suhlines. (A and B) RSI with 50 and 75 chromosomes. (C) 9 x 3 with 120 chromosomes. (D) 9 x 8 “h” f, with 150 chromosomes. (E) 5 x 2 with 230 chromosomes. x2300.
240
DEVELOPMENTAL
y7hPERCENT 601
1 l-l
VOLUME 34, 1973
BIOLOGY
1 PERCENT 3oc
I
,
I
I
I
I
160 200 NUMBER
240
9x6 “h” fg
5x2
Cf-iROMOSOME
FIG. 6. Chromosome numbers in polyploid cultures. Chromosome spreads were made of plasmodial nuclei of 9 x 8 “h” f,, 9 x 3, 4 x 3, and 5 x 2 and of sporulating nuclei of a heterokaryon of 5 x 2 and 9 x 8. Chromosomes were counted and the counts grouped in intervals of 10, then plotted vs frequency to give the histograms on the right side of the figure. On the left are nuclear diameters of interphase nuclei at 3-4 hr after mitosis. The number of spreads counted were, top to bottom, 44,42,63, and 30 of 5 x 2 and 23 of the 5 x 2-9 x 8 heterokaryon. Dashed line in the lower left panel is diameter of nuclei of the plasmodium grown from spores of the heterokaryon.
mitosis so that metaphase nuclei accumulate. (Both colchicine and Colcemid are ineffective in blocking metaphase in either the plasmodium or the amoeba of Physarum .) Once a better method for making amoeba chromosome spreads becomes available, it will be feasible to begin karyotyping because the number of chromosomes will be more manageable than in the plasmodium. Previous investigators have reported that the most frequent chromosome number in P. polycephalum, Wis 1 strain, is about 50 (Ross, 1966; Koevenig and Jackson, 1966), but that nuclei with fewer than 30 and more than 150 chromosomes can also occur in other isolates (Ross, 1966). Our findings are similar, except that we have found polyploids to be more prevalent
among Wis 1 derivatives than expected from the earlier papers. All of our most vigorously growing, readily sporulating Wis 1 derivatives contained nuclei with 50 and with 75 chromosomes, the larger nuclei making up 25 to 50% of the total. B173 x B174, which is derived from the “Indiana” isolate (Poulter and Dee, 1968), had 75 chromosomes in the majority of the nuclei, and several Wis 1 sublines had 150 or more chromosomes. Nuclei with fewer than 30 chromosomes were present in several sublines, particularly a x i, a Wis 1 derivative (Dee, 1962). We are now trying to clone a x i to get a culture further enriched for the small nuclei because they would be a satisfactory alternative to amoeba chromosomes for karyotyping. Furthermore, if the culture fruited, one might obtain amoe-
FIG. ‘7. Chromosome spreads of growing and sporulating plasmodia and of germinating spores. (A-C) M+zVII(IIe), growing and sporulating plasmodia and germinating spore. (D, E) a x i, growing and sporulating plasmodia. (F-H) 9 x 8, growing and sporulating plasmodia and germinating spore. (I, J) CL, two nuclei of growing plasmodium at left and one of sporulating plasmodium at right. All are at about x2300. 241
242
DEVELOPMENTAL BIOLOGY III,, GROWING
I,,, SPOR”LATINC
M3cVII (Ilel
--
60
40
20 I 40 NflCLEA4R
80
120
CHROMOSOME
80
N”titER
DIAMETER;4
FIG. 8. Chromosome numbers in growing and sporulating heterothallic plasmodia. Chromosome counts were made in nuclei of growing and sporulating plasmodia and data were plotted as for Fig. 4. Fifty spreads were counted for the growing 9 x 8 and sporulating M,cVII(IIe) plasmodia and 20 to 30 spreads for the other preparations. Diameters of nuclei in growing plasmodia at 3 to 4 hr after mitosis are on the left; chromosome counts for growing plasmodia are in the center, and those for sporangia are at the right.
bae with 12 to 15 chromosomes, and they could be expected to be much more sensitive to mutagens than are RSD 4 and other clones now being used by geneticists (Haugli and Dove, 1972; Dee, 1973). The Colonia homothallic strains did not seem to fit the same pattern as the heterothallic strains, for they had 35 to 40 chromosomes in most nuclei and polyploid nuclei were rare. It thus appears that both the homoand heterothallic strains will have to be karyotyped before it can be established whether the “true” diploid chromosome number is represented by the homothallic strains, by heterothallic nuclei with 50 to 55 chromosomes, or by heterothallic nuclei with 25-30 chromosomes. The polyploid nuclei in our cultures may have been caused by one or more of several
VOLUME 34, 1973
environmental factors acting on the amoebae or plasmodia. Heat is one of the more likely possibilities, since Brewer and Rusch (1968) found that heat shocking a plasmodium blocked mitosis but not DNA synthesis and E. Guttes has in fact used this treatment to make plasmodial cultures which are “8- and 16-ploid” (personal communication). High temperature probably has a similar effect on amoebae, since we found that warming spores before germination gave rise to a polyploid plasmodium. Another source of polyploid plasmodial nuclei might be multiple fusions of normal amoebae (Ross and Cummings, 1970). Since the percentage of polyploid nuclei was larger in new cultures than in those which had been growing in the laboratory for several years, it would seem that the polyploid nuclei arose during or soon after karyogamy, and not by transformation of
I”“““““” 80
1
GROWING
ISPORULATING!
40
PERCENT
t PERCENT
60
30
40
20
20
IO i
cot
PERCENT
60
30
4Ot
PERCENT
t
I
Ill
t
II
i--l 40
00
NUCLEAR CHROMOSOME NUMBER DIAMETER;fi FIG. 9. Chromosome numbers in growing and sporhomothallic plasmodia. ulating Chromosome spreads were made of growing and sporulating C50 and CL. The numbers of spreads counted for the growing plasmodia were 50 of C 50 and 25 of CL, and for sporangia, 25 of C 50 and 40 of CL.
et al. Chromosome
MOHBERC
Numbers
in Physarum
243
I PERCENT
B 173 x B 174
40 -
-y
GROWING
SPORULATING
-
3020 IO I
0
40
r
80
I
120
I
l-l
160
2000
CHROMOSOME
10. Chromosome the growing plasmodium FIG.
I
40
I
80
I
120
I
160
NUMBER
numbers in growing and sporulating B 173 x B 174. Forty spreads were counted for and 50 for precleavage sporangia. The histogram was plotted as for Fig. 6.
nuclei after the plasmodium had been established in axenic culture. The polyploid nuclei, at least those with about 75 chromosomes, did not seem to affect growth rate or ability to sporulate and the only objection to their presence seemed to be that it was difficult to study meiosis in cultures with mixed ploidy. The most highly polyploid sublines were useful for several purposes. For example, they were a better starting material for nucleolar isolation (Mohberg and Rusch, 1971) because they were uninucleolate and had proportionately larger nucleoli, and it was easier to break the nuclei and recover the nucleoli. Nuclear size made a convenient marker for following nuclei from two different plasmodia within a heterokaryon. Previous experiments with heterokaryons have involved fusion of plasmodia of the same subline but from different stages of the mitotic cycle (Rusch et al., 1966; Guttes and Guttes, 1968) or of the life cycle (Guttes and Guttes, 1969) in order to study the effects on timing of mitosis and DNA synthesis. It should be possible to use polyploid cultures in exactly the same way because (1) the ratio of whole plasmodial protein to DNA is constant, regardless of ploidy, and a given mass or volume of any subline should therefore contain the same amount of DNA (but not necessarily the same number of nuclei) as any other subline, and (2) nuclear surface area increases with DNA content so that the rate of DNA synthesis (Alfert and Das, 1969) would be
expected to be similar in nuclei of different ploidy levels. It should be pointed out that since our sublines do not all have the same plasmodial fusion type, certain heterokaryons cannot be made. This problem does not arise with the Guttes polyploids because they have the same fusion type as the plasmodium from which they are made and can be “back crossed” with the parent culture to give a heterokaryon with two sizes of nuclei. In the heterothallic sublines which sporulated and gave rapidly germinating spores we found that both the growing and sporulating plasmodia had nuclei with 50 and 75 chromosomes and the germinating spores had 20 to 25. Presumably a reductive division had occurred in the nuclei with 50 chromosomes, but we do not know whether the larger nuclei also participated and are now trying to obtain M,a or M,c cultures with single ploidy levels of 50 and 75 chromosomes so that we can study this further. Slow germination of spores of the homothallic strains prevented our counting chromosomes in germinating spores and we therefore have no direct evidence as to whether or not meiosis occurred. The fact that spores and plasmodial nuclei had the same DNA content supports that contention of von Stosch et al. (1964) that meiosis does not occur. However, our values for DNA content of the homothallic plasmodial nuclei may be low because we harvested plasmodia of all sublines at 3-6 hr after mitosis on the assumption that S
244
DEVELOPMENTAL BIOLOGY
phase occupied the first 3 hr after mitosis, as it does in M,c (Braun et al., 1965) and that nuclei were in G, phase with the 4C DNA content. If instead, the homothallic plasmodia resembled selfing Didymium iridis (Yemma and Therrien, 1972) and had a delayed S phase, mid-interphase nuclei might still have close to the 2C DNA content, thus making them appear to have the same ploidy as the spores, which are in haploid G, phase in heterothallic strains. When data on nuclear DNA content during sporulation (Arescaldino, 1971; Mohberg and Rusch, 1971) are related to the events occurring within the nucleus, it appears that a thorough study should be made of DNA synthesis during sporulation and spore maturation. It is quite certain that the sporangial nucleus enters the first meiotic division with the 4C DNA content. and that between day 1 and day 4 following cleavage the DNA content decreases to the 2 C level. This probably reflects digestion of nuclei in autophagic vacuoles (Aldrich, 1970; Charvat et al., 1972), since von Stosch et al. (1964) report that there are two meiotic divisions and that one nucleus of each division degenerates. Since the mature spore has the 2 C and not 1 C DNA content, it appears that the surviving nucleus duplicates its DNA by utilizing breakdown products from the other nuclei. There also would seem to be synthesis of DNA in amoebae immediately after the division accompanying spore germination, because they have the 2 C DNA content within a few hours of hatching. We should like to thank Dr. W. M. LeStourgeon for help with the photography, Mr. Homer Montague and Mr. Walter Fumosa for printing photographs, and Mrs. LaVila Winnie for technical assistance. REFERENCES ALDRICH, H. C. (1967). The ultrastructure of meiosis in three species of Physarum. Mycologia 59, 127-148. ALDRICY H. C. (1969). The ultrastructure of mitosis in myxamoebae and plasmodia of Physarum flauicomum. Amer. J. Bot. 56, 290-299. ALDRICH, H. C. (1970). Pre- and postmeiotic events in
VOLUME 34, 1973 spores of the myxomycete Didymium iridis. J Cell Biol. 47, 4A. ALDRICH, H. C., and CARROLL, G. (1971). Synaptonema1 complexes and meiosis in Didymium iridis: a reinvestigation. Mycologia 63, 308-316. ALFERT, M., and DAS, N. K. (1969). Evidence for control of the rate of nuclear DNA synthesis by the nuclear membrane in eukaryotic cells. Proc. Nut. Acad. Sci., U.S. 63, 123-128. ARESCALDINO, I. (1971). Evolution de la teneur en ADN des noyaux de Physarum polycephalum au tours de la sporulation. C. R. Acad. Sci. Ser. D 273, 398-401. BRAUN, R., MI’ITERMAYER, C., and RUSCH, H. P. (1965). Sequential temporal replication of DNA in Physarum polycephalum. Proc. Nat. Acad. Sci. U.S. 53, 924-931. BREWER, E. N., and Ruscq H. P. (1968). Effect of elevated temperature shocks on mitosis and on the initiation of DNA replication in Physarum polycephalum. Exp. Cell Res. 49, 79-86. BURTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CATHCART, M. E., and ALDRICH, H. C. (1972). A case of interrupted meiosis in Fuligo septica. Amer. J. Bot. 59, 665. CHARVAT, I., Ross, I., and CRONSHAW, J. (1972). Autophagy during differentiation in the slime mold, Perichaena uermicularis. J. Cell Biol. 55, 38a. DANIEL, J. W., and BALDWIN, H. H. (1964). Methods of culture for plasmodial myxomycetes. In “Methods in Cell Physiology” (D. M. Prescott, ed.), pp. 9941. Academic Press, New York. DANIEL, J. W., and Rusty H. P. (1962). Method for inducing sporulation of pure cultures of the myxomycete Physarum polycephalum. J. Bacterial. 83, 234-240.
DEE, J. (1962). Recombination in a myxomycete, Physarum polycephalum Schw. Genet. Res. 3, 11-23. DEE, J. (1966). Multiple alleles and other factors affecting plasmodium formation in the true slime mold Physarum polycephalum. J. Protozool. 13, 610-616. DEE, J. (1973). Aims and techniques of genetic analysis in Physarum polycephalum. Ber. Deut. Bot. Ges. in press. ELLISON, J. R., and BARR, H. J. (1972). Quinacrine fluorescence of specific chromosome regions. Chromosoma 36, 424-435. FELDMAN. M.. MELLO-SAMPAYO, T., and SEARS, E. R. (1966). Somatic association in Triticum aestiuum. Proc. Nat.Acad. Sci. U.S. 56,1192-1199. FULTON, C. (1970). Amebo-flagellates as research partners: the laboratory biology of Naegleria and Tetramitus. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. IV, pp. 341-476. GOODMAN, E. M. (1972). Axenic culture of myxamoe-
MOHBERG et al. Chromoso Ime Numbers bae of the myxomycete Physarum polycephalum. J. Bacterial. 111, 242-247. GUISES, E., and GU~ES, S. (1969). Initiation of mitosis in interphase plasmodia of Physarum polycephalum by coalescence with premitotic plasmodia. Experientia 25, 11681170. GUT~ES, E., GUT~ES, S., and RUSCH, H. P. (1961). Morphological observations on growth and differentiation of Physarum polycephalum grown in pure culture. Deuelop. Biol. 3, 588-614. GKJTTES, S., and GUTTES, E. (1968). Regulation of DNA replication in the nuclei of the slime mold Physarum polycephalum. Transplantation of nuclei by plasmodial coalescence. f. Cell Biol. 37,761-772. HAIJGL~ F. B. (1971). Mutagenesis, selection and genetic analysis in Physarum polycephalum. Ph.D. dissertation, University of Wisconsin. HAUGLI, F. B., and DOVE, W. F. (1972). Mutagenesis and mutant selection in Physarum polycephalum. Mol. Gen. Genet. 118, 109-124. HAUGLI, F. B., DOVE, W. F., and JIMENEZ, A. (1972). Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum. Mol. Gen. Genet. 118, 97-107. HOWARD, F. L. (1931). The life history of Physarum polycephalum. Amer. J. Bot. 18, 116132. KERR, S. J. (1967). A comparative study of mitosis in amoebae and plasmodia of the true slime mold Didymium nigripes. J. Protozool. 14,439-445. KERR, S. J. (1968). Ploidy level in the true slime mould Didymium nigripes. J. Gen. Microbial. 53,9-15. KOEVENIG, J. L., and JACKSON, R. C. (1966). Plasmodial mitoses and polyploidy in the myxomycete Physarum polycephalum. Mycologia 58, 662-667. LACOUR, L. (1941). Acetic-orcein: A new stain-fixative for chromosomes. Stain Technol. 16, 169-174. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCCORMICK, J. J., and NARDONE, R. M. (1970). The effect of nitrogen mustard on the nuclear cycle and DNA synthesis in Physarum polycephalum. Enp. Cell Res. 60, 247-256. MOHBERG, J., and RUSCH, H. P. (1969). Growth of the large plasmodia of the myxomycete Physarum polycephalum. J. Bacterial. 97, 1411-1418. MOHBERG, J., and RUSCH, H. P. (1971). Isolation and DNA content of nuclei of Phvsarum polycephalum. Exp. Cell Res. 66, 305-316. POULTER, R. T. M., and DEE, J. (1968). Segregation of factors controlling fusion between plasmodia of the
in Physarum
245
true slime mould, Physarum polycephalum. Genet. Res. 12, 71-79. PRESCOTT, D. M. (1956a). Relation between cell growth and cell division. II. The effect of cell size on cell growth rate and generation time in Amoebo proteus. Ezp. Cell Res. 11, 86-94. PRESCOTT, D. M. (1956b). Relation between cell growth and cell division. III. Changes in nuclear volume and growth rate and prevention of cell division in Amoeba proteus resulting from cytoplasmic amputation. Exp. Cell Res. 11, 94-98. REYNOLDS, E. S. (1968). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208212. Ross, I. K. (1966). Chromosome numbers in pure and gross cultures of myxomycetes. Amer. J. Bot. 53, 712-718. Ross, I. K., and CUMMINGS, R. 3. (1970). An unusual pattern of multiple cell and nuclear fusions in the heterothallic slime mold Didymium iridis. Protoplasma 70,281-294. RUSCH, H. P.. SACHSENMAIER,W., BEHRENS, K., and GRUTE~, V. (1966). Synchronization of mitosis by the fusion of the plasmodia of Physarum polycephalum. J. Cell Biol. 31, 204-209. SACHSENMAIER, W., and RUSCH, H. P. (1964). The effect of 5-fluoro-2’-deoxyuridine on synchronous mitosis in Physarum polycephalum. Exp. Cell Res. 36, 124-133. SACHSENMAIER, W., DONGES, K. H., RUPFF, H., and CZIHAK, G. (1970). Advanced initiation of synchronous mitoses in Physarum polycephalum following UV-irradiation. 2. Naturforsch. B 25,866-871. SUSSMAN, M., and SUSSMAN, R. R. (1962). Ploidal inheritance in Dictyostelium discoideum: stable haploid, stable diploid and metastable strains. J. Gen. Microbial. 28, 417-429. VON STOSCH, H. A. VAN ZUL-PISCHINGER, M., and DERSCH, G. (1964). Nuclear phase alternance in the myxomycete Physarum polycephalum. Abstr. 10th Int. Bot. Congr., pp. 481-482. WHEALS, A. E. (1970). A homothallic strain of the myxomycete Physarum polycephalum. Genetics 66, 623-633. YEMMA, J. J., and THERRIEN, C. D. (1972). Quantitative microspectrophotometry of nuclear DNA in selfing strains of the myxomycete, Didymium iridis. Amer. J. Bot. 59, 828-835. YOUNG, H. D. (1962). “Statistical Treatment of Experimental Data” pp. 101-126. McGraw-Hill, New York.
Nuclear
BIOLOGY
34, 228245
(1973)
DNA Content Myxomycete
and Chromosome Numbers Physarum polycephalum
in the
JOYCE MOHBERG, KARLEE L. BABCOCK, FINN B. HAUGLI, AND H. P. RUSCH McArdle
Laboratory,
Medical
School, University Accepted April
of Wisconsin, Madison,
Wisconsin 53706
20, 1973
An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum. It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein. Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20-25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35-40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein : DNA and RNA: DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker. A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined nlasmodial growth medium, containing 2% agar. Plasmodia are visible in 2-4 days. INTRODUCTION
The myxomycete, Physarum polycephaZum, would seem ideally suitable for biochemical studies of mitosis and meiosis and of haploid and diploid nuclei, since plasmodial mitosis and meiosis are both synchronous (Howard, 1931; Guttes et al., 1961; Aldrich, 1967; Arescaldino, 1971) and all stages of the life cycle can now be cultivated axenically (Daniel and Baldwin, 1964; Goodman, 1972). However, such investigations are meaningful only if the particular culture line under study actually undergoes meiosis, and it is clear that the mere fact that spores are formed does not prove that meiosis has occurred, since sporulation can occur without meiosis (von Stosch et al., 1964), meiosis I can occur without spore cleavage (Aldrich and Carroll, 1971) or meiosis I can be arrested until shortly before spore germination (Cathcart and Aldrich, 1972). In order to find a means of determining whether our cultures were completing meiosis, we did DNA analyses throughout the life cycle in sev-
era1 of our established sublines and found that those giving viable spores had twice the DNA content in the plasmodial nucleus as in the spore, whereas a culture giving nonviable spores had the same DNA content in the plasmodium as in the spores, and apparently did not undergo meiosis (Mohberg and Rusch, 1971). We now have extended the study to a series of sublines which have been growing in the laboratory for shorter periods of time, either because they had been kept as sclerotia since shortly after purification from field collections, or because they had arisen from recent matings of amoebae. This time we have done chromosome counts in plasmodia and germinating spores in addition to DNA analyses at different stages of the life cycle, and the data presented in this paper are consistent with the heterothallic strains being haploid in the amoeba and diploid in the plasmodium. Among the new matings of amoebae used in this study, several were found which had abnormally large nuclei. Al-
228 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
MOHBERC
et ~1. Chromosome
though most of them did not sporulate, these cultures were still of value because they provided an opportunity for studying nuclear: cytoplasmic relationships without subjecting the organism to such trauma as amputation (Prescott, 1956b) or exposure to visible (Prescott, 1956a) or ultraviolet light (Sachsenmaier et al., 1970). This paper gives data on chromosome numbers, on the relation of nuclear DNA and protein to nuclear volume and surface area, and on plasmodial protein : DNA and RNA : DNA ratios in normal and polyploid cultures. MATERIALS
AND
METHODS
Culture strains and methods. P. polycephalum amoebae clones RSD 2 through 9
DNA
CONTENT
OF PLASMODIAL
Numbers
were obtained by Rodger Steeper of this laboratory from spores of a plasmodium (RSI) grown from the Wis 1 isolate of Dr. M. Backus of the Botany Department of this university. Amoebae a, i, B173, B174 (Dee, 1962, 1966) and the homothallic (Wheals, 1970) strains C 5-1 and C 50 were the gift of Dr. J. Dee of the Genetics Department of the University of Leicester. Amoebae were grown on Escherichia coli lawns on agar or on Millipore membrane (Haugli, 1971) and were mated to give the plasmodia listed in Table 1. RSD amoeba clones were shown by Haugli (1971) to be of mating types 1 and 2 (Dee, 1962), and matings are referred to subsequently by number only, the mating type 1 parent
TABLE I NUCLEI AND OF SPORES OF DIFFERENT
Plasmodium pg per nucleus
7x 9x 5x M,ad axi ix B173 B173 x RSD8 B173 x B174 i x B174
SUBLINES
OF Physarum
Polycephalum
DNA Content
Subline
M,cVII M,cVII (Be) M,cIV c 5-1 c 50 RSP RSD4 x RSD 4x 5x 9x 4x 5x
229
in Physurum
6 8 6 8 3 3; 1st mating 2nd mating 3 3 2
1.23 1.35 1.15 1.07 0.88 0.97 1.21 1.34 1.13 1.04 1.92 1.88 1.87 1.87 1.84 3.64 0.91 0.94 1.18 1.16 1.77 1.94
* 0.08(4)” * 0.02(2) * 0.09(5)” f 0.03(6) + 0.03(5) * 0.03(4) * 0.14(4) * 0.02(3) f 0.08(7) * 0.07(6) * O.ll(5) + 0.05(5) * 0.17(5) * O.lO(5) zt 0.06(6) zt 0.19(6) * 0.09(4) * 0.05(7) f 0.01(2) f 0.04(2) * 0.13(5) * O.OO(2)
Spore pg per spore 0.62 + 0.03(5) 0.53 1.17 i 0.06(4) Nonsporulater 0.92 * O.OO(2) 0.65 * 0.06(4) Nonsporulater 1.09 * 0.10(2) Nonsporulater 0.60 i 0.04(5) Nonsporulater Nonsporulater Nonsporulater Nonsporulater Not analyzed Not analyzed 0.64 + 0.06(4) 0.74 * 0.01(2) Not analyzed Not analyzed Not analyzed
a Plasmodial nuclei were analyzed at 4-6 hr after mitosis and spores at l-2 weeks after cleavage, Nuclei and spores were washed and counted as described in Materials and Methods. DNA content is given in picograms per nucleus or spore + standard error; the number of preparations analyzed is given in parentheses, h From Mohberg and Rusch (1971). c Parent of RSD amoeba clones. d Parent of a and i amoeba clones.
230
DEVELOPMENTAL BIOLOGY
being given first. Other plasmodial sublines used were CL, a gift of J. Dee; Wis 2, a second strain collected by Dr. Backus; several M,‘s from an earlier purification of the Wis 1 isolate (Daniel and Baldwin, 1964); M,cVII(IIe), obtained by passing M,cVII through the life cycle several times (Haugli, 1971); 9 x 8 “h”, a possible homothallic obtained by Dr. D. N. Jacobson of this laboratory from spores of nitrosoguanidine-treated 9 x 8 plasmodium; and 9 x 8 “h” fz, a polyploid obtained by passing 9 x 8 “h” through the life cycle twice. Physarum flavicomum plasmodia were provided by Dr. W. M. LeStourgeon of this laboratory. Stock cultures of all plasmodia were carried as microplasmodia in submersed culture in semidefined medium (Daniel and Baldwin, 1964), and were stored as spherules. The M, sublines, RSI, a x i and the homothallic sublines were induced to spherulate by transferring microplasmodia to salts-citrate medium (Daniel and baldwin, 1964), but other sublines lysed in that solution and were therefore allowed to spherulate in exhausted nutrient medium as follows: Five milliliters of medium in 20 x 150-mm tubes were inoculated with microplasmodia and incubated for 10 days to 2 weeks on a gyratory shaker (New Brunswick, Model V with l-inch stroke) at 200 rpm at 23”-26°C. Tubes were then either stored’ at 4” or spherules were pelleted, washed with water, dried on filter paper, and stored in a desiccator at 4°C. Synchronous plasmodial cultures were grown in petri dishes on filters supported by stainless steel screen as described earlier (Mohberg and Rusch, 1969) except that Millipore membrane was used only for M, sublines and Schleicher and Schuell 576 filter paper was used for growing other sublines and for sporulating all sublines. Heterokaryons were made by inoculating dishes with equal amounts of microplasmodia of two sublines, as described by Haugli et al. (1972). Under these conditions all sublines fused initially but non-
VOLUME 34, 1973
compatible combinations-5 x 2 and M,cVII(IIe), for example-separated later, and sublines were therefore judged compatible only if they still gave a single disk-shaped plasmodium at 36 hr after inoculation. [See Haugli (1971) and Dee (1973), for plasmodial fusion characteristics of some M,c derivatives.] Sporulation was induced by illuminating plasmodia which had been starved on niacin-carbonate medium, either according to Daniel and Baldwin (1964) or to Haugli et al. (1972), or which had been starved on exhausted nutrient medium, made initially with 0.5 instead of 1% (w/v) glucose. The latter medium was always used with subline a x i because it sclerotized on the salts medium and was used with M, sublines whenever rapid spore germination was needed. Plasmodia were put into the illuminator (Daniel and Rusch, 1962) 4-7 days after inoculation and were illuminated for 4 hr each day until they sporulated. Spores were germinated by scraping sporangia (l-3 weeks after cleavage) into 40 ml of distilled water and crushing in a Potter-Elvehjem homogenizer with Teflon pestle. (Washing was done aseptically at room temperature.) The spores (25-75 million) were centrifuged at 1000 g for 5 min and washed three more times with water. They were then suspended in 5 ml of water and put into a petri dish. Germination in M,cVII(IIe) and 9 x 8 spores began within half an hour, with the highest proportion of metaphase figures occurring at 1 to 1.5 hr. CL and C 50 were slower with germination beginning in about 3 hr. To complete the life cycle, spores were left in water for 6 hr, by which time germination was finished, and l-ml aliquots were put into petri dishes containing 20 ml of one-tenth strength semidefined plasmodial growth medium (Daniel and Baldwin, 1964) in 2% agar. Spores were spread with a glass rod and the dishes were incubated at 26”. Alternatively, spores were put on Millipore membrane supported by a stainless steel grid
MOHBERC
et al. Chromosome
over diluted medium. With either system plasmodia were visible in 2-4 days. Chromosome counts. The mitotic stage of growing and sporulating plasmodia was determined by examining smears (Sachsenmaier and Rusch, 1964; McCormick and Nardone, 1970) with the phase contrast microscope. When nuclei of growing plasmodia entered prometaphase, cultures were cut in half and one half was immediately dipped in ice-water and homogenized (see below). The other half was harvested 2 min later. Since chromosomes spread well only during the middle 2-3 min of metaphase, 4-6 nuclear preparations were made to ensure that at least one would give usable spreads. Sporulating plasmodia were cut into fourths when they entered prophase and the segments were harvested at 15min intervals, since metaphase seemed to last longer than in the growing plasmodium and mitosis was not as tightly synchronous. Metaphase nuclei were isolated from growing plasmodia by slow homogenization in 40 ml of nuclear isolation medium (for details of equipment and medium composition, see Mohberg and Rusch, 1971). The suspension was passed through milk filter, divided equally between two conical centrifuge tubes and underlaid with 10 ml of 1 M sucrose solution. Tubes were centrifuged at 50 g for 15 min and the pellet was removed with a wide-tipped pipette. Nuclei were then pelleted by centrifuging at 1000 g for 15 min. After they had been washed once in 40 ml of nuclear isolation medium, they were suspended in 0.2 ml of the same solution by vortexing and were kept in ice while chromosome spreads were being made. Sporangia were washed with 0.25 M sucrose, containing 0.01 M ethylene diaminetetraacetate (pH 7.5) and were homogenized in 40 ml of nuclear isolation medium with 4 or 5 strokes in a Potter-Elvehjem homogenizer, followed by 2 min of slow stirring with a Waring Blendor. Nuclei were isolated from the homogenate as
Numbers
in Physarum
231
described for growing plasmodia except that they had to be passed through 1 M sucrose at least twice before they were sufficiently free of cytoplasmic granules (lipid and/or polyphosphate?) so that chromosomes would spread properly. Chromosome spreads were made by a modification of the method of LaCour (1941). A drop of nuclear suspension was mixed on a slide with 2 drops of lactic acid (2 parts of 88% lactic acid mixed with 1 part of water), and was heated over a small flame. (We are indebted to Dr. John Ellison, formerly of the Anatomy Department of this university, for this suggestion.) The slide was allowed to cool for several minutes, and 2 drops of 2% orcein in 75% acetic acid were added. The mixture was warmed again and covered with a cover slip. Five minutes later the preparation was flattened between several layers of paper towel. Observations and photographs were made with a phase contrast microscope within 12-24 hr. (Orcein stain was made by dissolving 2 gm of Baker synthetic orcein in 50 ml of boiling glacial acetic acid, and diluting the stock solution with 45% acetic acid immediately before use. The mixture was then centrifuged and filtered.) Analytical methods. Nuclei and spores were suspended in 0.25 M sucrose, containing 0.01 M CaCl,, and were counted in a hemacytometer or measured with a micrometer. An equal volume of 10% (w/v) trichloroacetic acid in acetone was then added to the nuclear suspension, and it was centrifuged at 75,000 g. The pellet was washed twice with 0.25 M perchloric acid at 0” and extracted twice with 0.5 M perchloric acid at 70”. DNA and protein were analyzed by the methods of Burton (1956) and Lowry et al. (1951) with the modifications described previously (Mohberg and Rusch, 1969). RESULTS
DNA Content of Plasmodial
Nuclei
Most of the sublines used in this study
232
DEVELOPMENTAL BIOLOGY
(Table 1) were “new”; that is, they had arisen from recent mating of amoebae or, as in the case of RSI and M ,cVIII, had been kept as spherules since shortly after purification from the Backus Wis 1 sclerotium. MscIV was included as an “old” culture because during 4 years of cultivation in the laboratory it had lost the ability to complete meiosis but not to sporulate (Mohberg and Rusch, 1971). M,a was included because we had two stocks, one (“new” M,a) which had been grown in the laboratory for a short time before it was stored as spherules (in 1962) and another (“old” M,a) which had been grown for several years before storage. We do not know which of these M,a’s is equivalent to Ross’s 51D (Ross, 1966). Nuclear DNA content (Table 1) was roughly 1 pg in the M, sublines, in most of the RSD and Dee amoeba crosses and in the homothallic strains C 5-1 and C 50. Diameter in these nuclei was between 3.0 and 3.5 pm. All matings of RSD 3 and of B174 had a DNA content of about 1.8 pg and a diameter of 4-4.5 pm. The one mating of RSD 2 which was analyzed had a diameter of about 6 pm and a DNA content of 3.6 pg. These sublines with higher nuclear DNA content grew somewhat slower than did M,c, but mitoses were synchronous and similar to those of normal cultures
except that nucleolar reconstruction was slower. The larger nuclei had larger nucleoli, and late interphase 9 x 3 and 5 x 2 had nucleolar diameters of approximately 2.5 and 3 pm, respectively, as compared to 1.8 pm in M,c. When the data of Table 1 were arranged as in Table 2, it appeared that the DNA content of amoebae RSD 4 through 9, provided that they mated 1 to 1, must have been about 0.6 pg, which agrees with the value of 0.55 pg found in nuclei of log phase RSD 4 and RSD 5 (Mohberg and Rusch, 1971). Amoebae RSD 3, however, appeared to contain 1.2 pg of DNA and RSD 2, 3 pg. Similarly, analyses of crosses of the Dee amoebae suggest that a, i, and B173 all contained 0.6 pg of DNA and B174 contained 1.2 pg like RSD 3. We have not done DNA analyses of any amoeba clones except 4 and 5 because, as reported before (Mohberg and Rusch, 1971), agar-grown cells cannot be used because the agar interferes with the diphenylamine reaction; and some clones, such as RSD 2 and 3, grew erratically on Millipore membrane. However, RSD 2 seemed definitely polyploid, judging from its large cell size and nuclear diameter, which was 3.8 pm, as compared to less than 3 in RSD 5. Sachsenmaier et al. (1970) have ob-
TABLE PREDICTED DNA Amoeba sublines mt, RSD 4
2
CONTENT OF NUCLEI OF RSD AMOEBAE” 8
DNA per amoeba nucleus ( pg)
0.6" 0.6 0.7 0.6
Amoeba sublines; mt2” RSD2
3
DNA per plasmodial -
6
nucleus (pg)
-
1.9 1.9 1.9 1.8
1.2 1.1 -
1.3 1.1
3.0
1.2
0.6
0.6
5
3.6
7 9 DNA per amoeba nuc-
VOLUME 34, 1973
leus (ps) a DNA content of plasmodia derived from RSD amoebae has been tabulated according to parent amoeba of each plasmodial subline, with mating type 2 parents across the top of the table and mating type 1 down the left side. The predicted DNA content of the mt, amoebae is in the right-hand column, and of the mt, amoebae across the bottom of the table. DThese values were obtained by analysis of isolated RSD 4 and 5 amoeba nuclei (Mohberg and Rusch, 1971).
MOHBERC et al. Chromosome
tained data from studies of UV-irradiated plasmodia which suggested that P. polycephalum resembles Amoeba proteus in having a critical cytoplasm : nucleus (protein : DNA) ratio (Prescott, 1956a, b) which must be reached before nuclear division can occur. It was therefore of interest to determine the protein : DNA and RNA: DNA ratios for whole microplasmodia of sublines with different nuclear DNA contents. Both protein : DNA and RNA : DNA ratios were roughly equal for all sublines tested (Table 3), indicating that those cultures with larger nuclei had proportionately fewer of them. Pigment : DNA ratios differed markedly among the various crosses and ranged from lemon yellow in 5 x 2 and 9 x 8 “h” f, to orange in 9 x 3 and 9 x 8. This was useful when microplasmodia of different sublines were mixed to make heterokaryons (see Materials and Methods) because noncompatible pairs, such as 5 x 2 and M,cVIII, could be distinguished both by the colors of the TABLE PROTEIN, RNA,
Numbers
233
in Physarum
plasmodia and by the size of the nuclei. In the myxamoebae of Dictyostelium discoideum (Sussman and Sussman, 1962) and in the amoeba Naegleria (Fulton, 1970), nuclear volume is proportional to DNA content. On the other hand, the rate of DNA synthesis in plant and animal cells appears to be related to nuclear surface area (Alfert and Das, 1969). We therefore plotted the surface area and volume of nuclei of different sublines against both DNA and protein content. Figure 1 shows that both DNA and protein content gave essentially straight lines when plotted against both volume and surface area. However, only DNA vs surface area passed through the origin. The other three curves all intercepted the y axis. DNA Content of Spores
Most of the sublines with a nuclear DNA content of about 1 pg sporulated, and all but 4 x 8, C 50 and M,cIV gave spores which contained 0.5 to 0.6 pg of DNA when 3
AND PIGMENT CONTENT OF MICROPLASMODIA OF SUBLINES WITH DIFFERING NUCLEAR DNA CONTENT
Subline
M,cIV axi 9x8 Mean B173 x B174 i x B174 4x3 9x3 9 x 8 “b” f, Mean 5 x 2; 1 yr in culture 5x2
DNA per nucleus (PP)
Composition Protein/DNA ~ccdrg)
of microplasmodia” RNA/DNA (KdPP)
1.2 1.0 1.1
85.9 85.3 81.8
13.3 13.0 10.8
1.1
84.3
12.4
1.8 1.9 1.9 1.8 2.0
70.5 70.5 91.3 78.4 84.7
10.8 Not done 13.4 12.0 9.8
1.9
79.1
11.5
2.7 3.6
92.1 75.5
13.7 11.7
PigmentiDNAD 0.24 0.55 0.62
0.46 0.54 0.67 0.81 0.30
0.26 0.29
a Microplasmodia in early to mid log phase growth were pelleted, extracted with TCA-acetone to remove pigment, and were washed with cold perchloric acid and extracted with hot PCA as described earlier (Mohberg and Rusch, 1969). Each value represents the mean of analyses of two to four samples. b Pigment is expressed here as optical density at 415 nm of 1 ml of TCA-acetone extract in a 13-mm light path (Daniel and Baldwin, 1964).
234
DEVELOPMENTAL BIOLOGY
VOLUME 34, 1973
B174 were ever observed to fruit. DNA analyses were not done on spores of either subline but chromosome counts were made in B173 x B174 during sporulation. Results are in the following section. Chromosome Plasmodia
0x1 -t-
0
I
I
40 80 VOLUME;
I
120 p3
I
0
I
I
40 80 120 SURFACE AREA; ~~
FIG. 1. Relation of nuclear volume and surface area to DNA and protein content. Nuclear diameters of plasmodial nuclei at 3 hr after mitosis were plotted vs frequency, as in Figs. 6 and 8. Nuclear volumes and surface areas were calculated from the areas under the diameter curves and were plotted vs DNA and protein content in picograms per nucleus. Curves were fitted by the method of least squares (Young, 1962). Points for normal-sized nuclei are labeled in the right-hand panels and points for 5 x 2 and the matings of amoeba RSD 3 with RSD 4, 5, 7, and 9 are labeled in the left-hand panels.
mature. (Germination in 4 x 8 and C 50 was 10-15s and in M,cIV, less than 0.1s.) Table 4 shows that the DNA content of spores remained at about 1 pg per spore for the first 24 hr after cleavage, and then slowly decreased during the next several days to a final value of about 0.6 pg, where other experiments showed that it remained for at least the next 3 weeks. Spores were capable of germinating by 4 days after cleavage, and swarm cells began to emerge within 30 min of plating. Germination was complete in about 3 hr. Amoebae divided before, during and after spore germination so that by 5 hr after plating there were two amoebae for each spore that had hatched. At this time the myxamoebae from 9 x 8, the only subline yet analyzed, contained 0.55 pg of DNA per nucleus. Of the sublines with a DNA content of 1.8 pg and above, only 5 x 2 and B 173 x
Counts
in
Growing
Ross (1966) and Kerr (1968) both reported having considerable difficulty in making chromosome spreads with whole slime mold plasmodium. Our own earlier efforts showed that the cytoplasm not only kept the nuclei from flattening adequately, but also seemed to interfere with staining of the chromosomes. We therefore tried using isolated metaphase nuclei as starting material because mitosis is intranuclear (Figs. 2A and 2B), and it should therefore be possible to get rid of cytoplasm without losing chromosomes. This proved successful, provided that nuclei were heated in lactic acid to spread the chromosomes before stain was added. Germinating spores and growing amoebae were not fracTABLE
4
EFFECT OF AGE OF SPORESON THEIR DNA CONTENTS Age of spores (hr post-cleavage)*
Sublines” 9 x 8’
M,cVII(IIe)’
DNA content (pg per spore)
12 to
1 4 24 48 72 96 144 15 days
1.11 Not done 0.86 0.80 Not done 0.79 0.68 0.60 + 0.04(5)
Not done 0.94 0.91 Not done 0.63 Not done 0.67 0.53
o Spores of 9 x 8 and of M,cVII(IIe) were produced in niacin-salts medium and harvested at the times indicated. Spores were washed, counted, and analyzed as described in Materials and Methods. b Mitosis occurred at 13 hr after the end of illumination, spore cleavage 0.5 to 1 hr after mitosis, and melanization of spores at about 1 hr after cleavage. c DNA content of plasmodial nuclei (Table 1) was 1.04 * 0.07(6) in 9 x 8 and 1.35 + 0.02(Z) in M,cVII(IIe).
MOHBERG
et al. Chromosome
Numbers
in Physarum
235
FIG. 2. Photomicrographs of starting material for chromosome spreads. (A) Isolated metaphase nuclei of growing plasmodia of M,cIV (left) and of 5 x 2 (center) and of a sporulating plasmodium of M,cVIII (right). Nuclei were isolated as described earlier (Mohberg and Rusch, 1971) and were stained with acetic-orcein and photographed with phase contrast. (B) Electron micrograph of isolated metaphase nuclei of a growing M,cIV plasmodium. Isolated nuclei were fixed with glutaraldehyde and stained with osmic acid and lead, according to Reynolds (1968). (C) Germinating spores of M,cVII(IIe), about 2 hr after they were plated as described in Materials and Methods. Phase contrast: unstained. (D) Higher magnification of metaphase in a germinating spore. Acetic-orcein stained; phase contrast. A and D, x2300; B, ~22,000; C, x500.
236
DEVELOPMENTAL BIOLOGY
tionated but were spread by heating the whole cells in lactic acid, since the nuclear membrane disappears during mitosis in the amoeba (Kerr, 1967; Aldrich, 1969). Chromosome spreads of nuclei from different stages of the life cycle are shown in Figs. 3 and 7. In the growing plasmodium most chromosomes were very similar in size and shape, but there were some distinctively different chromosomes, such as the very long ones at 4 to 5 o’clock in Fig. 3A. Banding along the chromosomes was evident in many spreads, and it was expected that certain of these bands might be stained with quinacrine (Ellison and Barr, 1971), but Dr. Ellison (personal communication) was unable to find any quinacrine-positive regions in our preparations. Somatic association (Feldman et al., 1966) could be seen in spreads of both growing (Figs. 3A and 3B) and sporulating plasmodia. In late metaphase, chromosomes sometimes could be spread sufficiently thin so that individual chromatids could be seen (Figs. 3C and 7G). It is obvious from Fig. 3 that the number of chromosomes per spread differed markedly, even among sublines a x i, M,a, and M,c, all of which had similar nuclear DNA contents. Histograms of chromosome number (Figs. 4 and 8) revealed that chromosome number varied as much within a subline as among sublines. In “old” M,a and M,cIV, both of which had been growing in the laboratory for a long time, most contained 50-55 chromosomes, nuclei similar to Ross’s 51D (Ross, 1966). In RSI (Figs. 4 and 5B) and “new” M,a (not shown) about 25%, and in M,cVIII and M,cVII(IIe) (Figs. 7A and 8) almost 50%, of the nuclei had 75 or more chromosomes. On the other hand, in a x i 25% of the nuclei had 25-30 chromosomes (Figs. 3A and 4), and in the homothallic cultures essentially all nuclei had 35-40 chromosomes (Figs. 4 and 9). In P. flauicomum (not shown) the major peak was between 80 and 90 chromosomes, which is somewhat higher than the 70 found by Ross (1966),
VOLUME 34, 1973
and there were smaller peaks at about 50, 70, and 100. Among the sublines with a higher nuclear DNA content, 9 x 8 “h” f, had two ploidy levels, one with 150 (Fig. 5D) and one with 175 chromosomes (Fig. 6). In 4 x 3 there were peaks at 75, 100, and 150 chromosomes and in 9 x 3 at 75 and 100 (Fig. 5C). In 5 x 2 there were between 150 and 240 chromosomes in the majority of the nuclei. This probably is owing both to the presence of several ploidy levels and the difficulty of flattening very large nuclei sufficiently for all chromosomes to be seen without at the same time stretching and breaking others. Both 9 x 3 and 5 x 2 contained a few nuclei with 50 chromosomes, and these presumably survive better under adverse conditions than do the larger nuclei, thus resulting in the occasional drop in nuclear size that was seen with polyploid cultures (see later section). Chromosome
Counts in Sporangia
Although both M,cVIII and RSI fruited readily, chromosome counts on the precleavage nuclear division were done with sublines M,cVII(IIe), 9 x 8 and 9 x 8 “h,” since the spores of these cultures gave 50-60% germination, as compared to 5-10% in M,cVIII and RSI, and it could therefore be assumed that at least half of the nuclei which divided before spore cleavage went on to complete meiosis. Since a x i and the Colonia strains have been used frequently in genetic studies (Wheals, 1971; Dee, 1973), these cultures also were included, although the spore germination was too slow to permit counting chromosomes in hatching amoebae. In both the heterothallic (Fig. 8) and the homothallic (Fig. 9) strains the same ploidy levels were seen in the sporangium as in the growing plasmodium. Germinating spores of “new” M 3a, M,cVII(IIe), 9 x 8, and 9 x 8 “h” had 20-25 chromosomes, indicating that reduction had occurred at some time between spore cleavage and spore germination.
MOHBERG et al. Chromosome
Numbers
in Physarum
237
FIG. 3. Photomicrographs of representative chromosome spreads. Metaphase nuclei were from growing plasmodia for all spreads except H and I. (A) a x i, showing individual chromosomes. (B) 4 x 3, showing somatic association. (C) 9 x 8 “h” f,, showing splitting chromosomes. (D) M,a (Ross’s 5lD), 47 chromosomes. (E) P. fkxuicomum, 86 chromosomes. (F) C 50, 40 chromosomes. (G) M,cVIII, 80 chromosomes. (H) M,cVII(IIe) germinating spore, 22 chromosomes. (I) Growing amoeba from spores of the 5 x 2-9 x 8 heterokaryon, 30 chromosomes. All spreads are at x 2300.
238
DEVELOPMENTAL BIOLOGY
VOLUME 34, 1973
Factors Affecting
FIG. 4. Chromosome numbers in representative plasmodial sublines. Chromosome spreads were made of growing plasmodia of the sublines indicated on the graph. Chromosomes were counted for a number of spreads for each subline, and counts were grouped into intervals of 5. A histogram was then made with frequency, as percent of total spreads counted, vs chromosome number. The number of spreads counted were70ofM,cVIIandC5-1,50ofa x i,40ofM+and RSI, and 25 of M,cIV.
As mentioned above, the only polyploid subline from Table 1 which fruited readily was B173 x B174, which had about 75 chromosomes during growth and sporulation (Fig. 10). Spores germinated too slowly for counts to be made in hatching amoebae. Since 5 x 2 could no longer be induced to sporulate after the method for counting chromosomes had been worked out, we resorted to making a heterokaryon with 9 x 8, which fruited very readily. In the precleavage mitosis (Fig. 6, dashed lines in bottom panel) the majority of the nuclei had 50 and 75 chromosomes like the 9 x 8 parent, and only a small fraction had the ploidy of the 5 x 2 parent. Moreover, the nuclear diameter in the plasmodium which arose from the heterokaryotic spores was about 3.5 pm, the same as in 9 x 8. Similar results were obtained when a 9 x 3-9 x 8 heterokaryon fruited. Recently we have obtained a derivative of M,cVII(IIe) which has 100-110 chromosomes and sporulates readily, giving polyploid amoebae. This culture was grown from spores which were heated to 37” for 10 min during the washing procedure (see Materials and Methods.)
Ploidy
On several occasions nuclear size of cultures dropped spontaneously. One example was a 5 x 2 culture which during a year of cultivation dropped from 3.6 to 2.7 pg of DNA per nucleus. Other examples were 5 x 2, 7 x 3, and B173 x 174 cultures which had normal-sized nuclei after they were freed of an unidentified contaminant by migrating across water agar. Results in the preceding section suggested that sporulation might select against polyploid nuclei. When B173 x 174 was passed through spores, the resulting plasmodium had a higher percentage of nuclei with 50 chromosomes, but when M,cVII(IIe) was handled similarly, the percentage of nuclei with 75 chromosomes did not decrease., Recloning amoebae before mating also was ineffective in reducing the fraction of polyploid nuclei. As mentioned above, an increase in ploidy was obtained when spores were heated before germination. DISCUSSION
The main problem in making chromosome spreads of isolated metaphase nuclei of the synchronous plasmodium of P. polycephalum is that metaphase lasts only about 7 min (Mohberg and Rusch, 1971), and it is therefore difficult to judge harvesting time so that nuclei are arrested in mid-metaphase when chromosomes are condensed and before they split into chromatids. However, once a good preparation has been made, it gives dozens of usable spreads on a single slide. Myxamoebae are much more difficult to work with because cultures do not have natural synchrony and metaphase is of about the same duration as in the plasmodium, so that even very rapidly proliferating cultures have only a small percentage of nuclei in the portion of metaphase giving good spreads. It appears that before reliable data can be obtained it will first be necessary to devise a technique either to synchronize amoebae or to block
MOHBERC et al. Chromosome
Numbers
in Physarum
239
FIG. 5. Chromosome spreads of polyploid suhlines. (A and B) RSI with 50 and 75 chromosomes. (C) 9 x 3 with 120 chromosomes. (D) 9 x 8 “h” f, with 150 chromosomes. (E) 5 x 2 with 230 chromosomes. x2300.
240
DEVELOPMENTAL
y7hPERCENT 601
1 l-l
VOLUME 34, 1973
BIOLOGY
1 PERCENT 3oc
I
,
I
I
I
I
160 200 NUMBER
240
9x6 “h” fg
5x2
Cf-iROMOSOME
FIG. 6. Chromosome numbers in polyploid cultures. Chromosome spreads were made of plasmodial nuclei of 9 x 8 “h” f,, 9 x 3, 4 x 3, and 5 x 2 and of sporulating nuclei of a heterokaryon of 5 x 2 and 9 x 8. Chromosomes were counted and the counts grouped in intervals of 10, then plotted vs frequency to give the histograms on the right side of the figure. On the left are nuclear diameters of interphase nuclei at 3-4 hr after mitosis. The number of spreads counted were, top to bottom, 44,42,63, and 30 of 5 x 2 and 23 of the 5 x 2-9 x 8 heterokaryon. Dashed line in the lower left panel is diameter of nuclei of the plasmodium grown from spores of the heterokaryon.
mitosis so that metaphase nuclei accumulate. (Both colchicine and Colcemid are ineffective in blocking metaphase in either the plasmodium or the amoeba of Physarum .) Once a better method for making amoeba chromosome spreads becomes available, it will be feasible to begin karyotyping because the number of chromosomes will be more manageable than in the plasmodium. Previous investigators have reported that the most frequent chromosome number in P. polycephalum, Wis 1 strain, is about 50 (Ross, 1966; Koevenig and Jackson, 1966), but that nuclei with fewer than 30 and more than 150 chromosomes can also occur in other isolates (Ross, 1966). Our findings are similar, except that we have found polyploids to be more prevalent
among Wis 1 derivatives than expected from the earlier papers. All of our most vigorously growing, readily sporulating Wis 1 derivatives contained nuclei with 50 and with 75 chromosomes, the larger nuclei making up 25 to 50% of the total. B173 x B174, which is derived from the “Indiana” isolate (Poulter and Dee, 1968), had 75 chromosomes in the majority of the nuclei, and several Wis 1 sublines had 150 or more chromosomes. Nuclei with fewer than 30 chromosomes were present in several sublines, particularly a x i, a Wis 1 derivative (Dee, 1962). We are now trying to clone a x i to get a culture further enriched for the small nuclei because they would be a satisfactory alternative to amoeba chromosomes for karyotyping. Furthermore, if the culture fruited, one might obtain amoe-
FIG. ‘7. Chromosome spreads of growing and sporulating plasmodia and of germinating spores. (A-C) M+zVII(IIe), growing and sporulating plasmodia and germinating spore. (D, E) a x i, growing and sporulating plasmodia. (F-H) 9 x 8, growing and sporulating plasmodia and germinating spore. (I, J) CL, two nuclei of growing plasmodium at left and one of sporulating plasmodium at right. All are at about x2300. 241
242
DEVELOPMENTAL BIOLOGY III,, GROWING
I,,, SPOR”LATINC
M3cVII (Ilel
--
60
40
20 I 40 NflCLEA4R
80
120
CHROMOSOME
80
N”titER
DIAMETER;4
FIG. 8. Chromosome numbers in growing and sporulating heterothallic plasmodia. Chromosome counts were made in nuclei of growing and sporulating plasmodia and data were plotted as for Fig. 4. Fifty spreads were counted for the growing 9 x 8 and sporulating M,cVII(IIe) plasmodia and 20 to 30 spreads for the other preparations. Diameters of nuclei in growing plasmodia at 3 to 4 hr after mitosis are on the left; chromosome counts for growing plasmodia are in the center, and those for sporangia are at the right.
bae with 12 to 15 chromosomes, and they could be expected to be much more sensitive to mutagens than are RSD 4 and other clones now being used by geneticists (Haugli and Dove, 1972; Dee, 1973). The Colonia homothallic strains did not seem to fit the same pattern as the heterothallic strains, for they had 35 to 40 chromosomes in most nuclei and polyploid nuclei were rare. It thus appears that both the homoand heterothallic strains will have to be karyotyped before it can be established whether the “true” diploid chromosome number is represented by the homothallic strains, by heterothallic nuclei with 50 to 55 chromosomes, or by heterothallic nuclei with 25-30 chromosomes. The polyploid nuclei in our cultures may have been caused by one or more of several
VOLUME 34, 1973
environmental factors acting on the amoebae or plasmodia. Heat is one of the more likely possibilities, since Brewer and Rusch (1968) found that heat shocking a plasmodium blocked mitosis but not DNA synthesis and E. Guttes has in fact used this treatment to make plasmodial cultures which are “8- and 16-ploid” (personal communication). High temperature probably has a similar effect on amoebae, since we found that warming spores before germination gave rise to a polyploid plasmodium. Another source of polyploid plasmodial nuclei might be multiple fusions of normal amoebae (Ross and Cummings, 1970). Since the percentage of polyploid nuclei was larger in new cultures than in those which had been growing in the laboratory for several years, it would seem that the polyploid nuclei arose during or soon after karyogamy, and not by transformation of
I”“““““” 80
1
GROWING
ISPORULATING!
40
PERCENT
t PERCENT
60
30
40
20
20
IO i
cot
PERCENT
60
30
4Ot
PERCENT
t
I
Ill
t
II
i--l 40
00
NUCLEAR CHROMOSOME NUMBER DIAMETER;fi FIG. 9. Chromosome numbers in growing and sporhomothallic plasmodia. ulating Chromosome spreads were made of growing and sporulating C50 and CL. The numbers of spreads counted for the growing plasmodia were 50 of C 50 and 25 of CL, and for sporangia, 25 of C 50 and 40 of CL.
et al. Chromosome
MOHBERC
Numbers
in Physarum
243
I PERCENT
B 173 x B 174
40 -
-y
GROWING
SPORULATING
-
3020 IO I
0
40
r
80
I
120
I
l-l
160
2000
CHROMOSOME
10. Chromosome the growing plasmodium FIG.
I
40
I
80
I
120
I
160
NUMBER
numbers in growing and sporulating B 173 x B 174. Forty spreads were counted for and 50 for precleavage sporangia. The histogram was plotted as for Fig. 6.
nuclei after the plasmodium had been established in axenic culture. The polyploid nuclei, at least those with about 75 chromosomes, did not seem to affect growth rate or ability to sporulate and the only objection to their presence seemed to be that it was difficult to study meiosis in cultures with mixed ploidy. The most highly polyploid sublines were useful for several purposes. For example, they were a better starting material for nucleolar isolation (Mohberg and Rusch, 1971) because they were uninucleolate and had proportionately larger nucleoli, and it was easier to break the nuclei and recover the nucleoli. Nuclear size made a convenient marker for following nuclei from two different plasmodia within a heterokaryon. Previous experiments with heterokaryons have involved fusion of plasmodia of the same subline but from different stages of the mitotic cycle (Rusch et al., 1966; Guttes and Guttes, 1968) or of the life cycle (Guttes and Guttes, 1969) in order to study the effects on timing of mitosis and DNA synthesis. It should be possible to use polyploid cultures in exactly the same way because (1) the ratio of whole plasmodial protein to DNA is constant, regardless of ploidy, and a given mass or volume of any subline should therefore contain the same amount of DNA (but not necessarily the same number of nuclei) as any other subline, and (2) nuclear surface area increases with DNA content so that the rate of DNA synthesis (Alfert and Das, 1969) would be
expected to be similar in nuclei of different ploidy levels. It should be pointed out that since our sublines do not all have the same plasmodial fusion type, certain heterokaryons cannot be made. This problem does not arise with the Guttes polyploids because they have the same fusion type as the plasmodium from which they are made and can be “back crossed” with the parent culture to give a heterokaryon with two sizes of nuclei. In the heterothallic sublines which sporulated and gave rapidly germinating spores we found that both the growing and sporulating plasmodia had nuclei with 50 and 75 chromosomes and the germinating spores had 20 to 25. Presumably a reductive division had occurred in the nuclei with 50 chromosomes, but we do not know whether the larger nuclei also participated and are now trying to obtain M,a or M,c cultures with single ploidy levels of 50 and 75 chromosomes so that we can study this further. Slow germination of spores of the homothallic strains prevented our counting chromosomes in germinating spores and we therefore have no direct evidence as to whether or not meiosis occurred. The fact that spores and plasmodial nuclei had the same DNA content supports that contention of von Stosch et al. (1964) that meiosis does not occur. However, our values for DNA content of the homothallic plasmodial nuclei may be low because we harvested plasmodia of all sublines at 3-6 hr after mitosis on the assumption that S
244
DEVELOPMENTAL BIOLOGY
phase occupied the first 3 hr after mitosis, as it does in M,c (Braun et al., 1965) and that nuclei were in G, phase with the 4C DNA content. If instead, the homothallic plasmodia resembled selfing Didymium iridis (Yemma and Therrien, 1972) and had a delayed S phase, mid-interphase nuclei might still have close to the 2C DNA content, thus making them appear to have the same ploidy as the spores, which are in haploid G, phase in heterothallic strains. When data on nuclear DNA content during sporulation (Arescaldino, 1971; Mohberg and Rusch, 1971) are related to the events occurring within the nucleus, it appears that a thorough study should be made of DNA synthesis during sporulation and spore maturation. It is quite certain that the sporangial nucleus enters the first meiotic division with the 4C DNA content. and that between day 1 and day 4 following cleavage the DNA content decreases to the 2 C level. This probably reflects digestion of nuclei in autophagic vacuoles (Aldrich, 1970; Charvat et al., 1972), since von Stosch et al. (1964) report that there are two meiotic divisions and that one nucleus of each division degenerates. Since the mature spore has the 2 C and not 1 C DNA content, it appears that the surviving nucleus duplicates its DNA by utilizing breakdown products from the other nuclei. There also would seem to be synthesis of DNA in amoebae immediately after the division accompanying spore germination, because they have the 2 C DNA content within a few hours of hatching. We should like to thank Dr. W. M. LeStourgeon for help with the photography, Mr. Homer Montague and Mr. Walter Fumosa for printing photographs, and Mrs. LaVila Winnie for technical assistance. REFERENCES ALDRICH, H. C. (1967). The ultrastructure of meiosis in three species of Physarum. Mycologia 59, 127-148. ALDRICY H. C. (1969). The ultrastructure of mitosis in myxamoebae and plasmodia of Physarum flauicomum. Amer. J. Bot. 56, 290-299. ALDRICH, H. C. (1970). Pre- and postmeiotic events in
VOLUME 34, 1973 spores of the myxomycete Didymium iridis. J Cell Biol. 47, 4A. ALDRICH, H. C., and CARROLL, G. (1971). Synaptonema1 complexes and meiosis in Didymium iridis: a reinvestigation. Mycologia 63, 308-316. ALFERT, M., and DAS, N. K. (1969). Evidence for control of the rate of nuclear DNA synthesis by the nuclear membrane in eukaryotic cells. Proc. Nut. Acad. Sci., U.S. 63, 123-128. ARESCALDINO, I. (1971). Evolution de la teneur en ADN des noyaux de Physarum polycephalum au tours de la sporulation. C. R. Acad. Sci. Ser. D 273, 398-401. BRAUN, R., MI’ITERMAYER, C., and RUSCH, H. P. (1965). Sequential temporal replication of DNA in Physarum polycephalum. Proc. Nat. Acad. Sci. U.S. 53, 924-931. BREWER, E. N., and Ruscq H. P. (1968). Effect of elevated temperature shocks on mitosis and on the initiation of DNA replication in Physarum polycephalum. Exp. Cell Res. 49, 79-86. BURTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CATHCART, M. E., and ALDRICH, H. C. (1972). A case of interrupted meiosis in Fuligo septica. Amer. J. Bot. 59, 665. CHARVAT, I., Ross, I., and CRONSHAW, J. (1972). Autophagy during differentiation in the slime mold, Perichaena uermicularis. J. Cell Biol. 55, 38a. DANIEL, J. W., and BALDWIN, H. H. (1964). Methods of culture for plasmodial myxomycetes. In “Methods in Cell Physiology” (D. M. Prescott, ed.), pp. 9941. Academic Press, New York. DANIEL, J. W., and Rusty H. P. (1962). Method for inducing sporulation of pure cultures of the myxomycete Physarum polycephalum. J. Bacterial. 83, 234-240.
DEE, J. (1962). Recombination in a myxomycete, Physarum polycephalum Schw. Genet. Res. 3, 11-23. DEE, J. (1966). Multiple alleles and other factors affecting plasmodium formation in the true slime mold Physarum polycephalum. J. Protozool. 13, 610-616. DEE, J. (1973). Aims and techniques of genetic analysis in Physarum polycephalum. Ber. Deut. Bot. Ges. in press. ELLISON, J. R., and BARR, H. J. (1972). Quinacrine fluorescence of specific chromosome regions. Chromosoma 36, 424-435. FELDMAN. M.. MELLO-SAMPAYO, T., and SEARS, E. R. (1966). Somatic association in Triticum aestiuum. Proc. Nat.Acad. Sci. U.S. 56,1192-1199. FULTON, C. (1970). Amebo-flagellates as research partners: the laboratory biology of Naegleria and Tetramitus. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. IV, pp. 341-476. GOODMAN, E. M. (1972). Axenic culture of myxamoe-
MOHBERG et al. Chromoso Ime Numbers bae of the myxomycete Physarum polycephalum. J. Bacterial. 111, 242-247. GUISES, E., and GU~ES, S. (1969). Initiation of mitosis in interphase plasmodia of Physarum polycephalum by coalescence with premitotic plasmodia. Experientia 25, 11681170. GUT~ES, E., GUT~ES, S., and RUSCH, H. P. (1961). Morphological observations on growth and differentiation of Physarum polycephalum grown in pure culture. Deuelop. Biol. 3, 588-614. GKJTTES, S., and GUTTES, E. (1968). Regulation of DNA replication in the nuclei of the slime mold Physarum polycephalum. Transplantation of nuclei by plasmodial coalescence. f. Cell Biol. 37,761-772. HAIJGL~ F. B. (1971). Mutagenesis, selection and genetic analysis in Physarum polycephalum. Ph.D. dissertation, University of Wisconsin. HAUGLI, F. B., and DOVE, W. F. (1972). Mutagenesis and mutant selection in Physarum polycephalum. Mol. Gen. Genet. 118, 109-124. HAUGLI, F. B., DOVE, W. F., and JIMENEZ, A. (1972). Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum. Mol. Gen. Genet. 118, 97-107. HOWARD, F. L. (1931). The life history of Physarum polycephalum. Amer. J. Bot. 18, 116132. KERR, S. J. (1967). A comparative study of mitosis in amoebae and plasmodia of the true slime mold Didymium nigripes. J. Protozool. 14,439-445. KERR, S. J. (1968). Ploidy level in the true slime mould Didymium nigripes. J. Gen. Microbial. 53,9-15. KOEVENIG, J. L., and JACKSON, R. C. (1966). Plasmodial mitoses and polyploidy in the myxomycete Physarum polycephalum. Mycologia 58, 662-667. LACOUR, L. (1941). Acetic-orcein: A new stain-fixative for chromosomes. Stain Technol. 16, 169-174. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCCORMICK, J. J., and NARDONE, R. M. (1970). The effect of nitrogen mustard on the nuclear cycle and DNA synthesis in Physarum polycephalum. Enp. Cell Res. 60, 247-256. MOHBERG, J., and RUSCH, H. P. (1969). Growth of the large plasmodia of the myxomycete Physarum polycephalum. J. Bacterial. 97, 1411-1418. MOHBERG, J., and RUSCH, H. P. (1971). Isolation and DNA content of nuclei of Phvsarum polycephalum. Exp. Cell Res. 66, 305-316. POULTER, R. T. M., and DEE, J. (1968). Segregation of factors controlling fusion between plasmodia of the
in Physarum
245
true slime mould, Physarum polycephalum. Genet. Res. 12, 71-79. PRESCOTT, D. M. (1956a). Relation between cell growth and cell division. II. The effect of cell size on cell growth rate and generation time in Amoebo proteus. Ezp. Cell Res. 11, 86-94. PRESCOTT, D. M. (1956b). Relation between cell growth and cell division. III. Changes in nuclear volume and growth rate and prevention of cell division in Amoeba proteus resulting from cytoplasmic amputation. Exp. Cell Res. 11, 94-98. REYNOLDS, E. S. (1968). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208212. Ross, I. K. (1966). Chromosome numbers in pure and gross cultures of myxomycetes. Amer. J. Bot. 53, 712-718. Ross, I. K., and CUMMINGS, R. 3. (1970). An unusual pattern of multiple cell and nuclear fusions in the heterothallic slime mold Didymium iridis. Protoplasma 70,281-294. RUSCH, H. P.. SACHSENMAIER,W., BEHRENS, K., and GRUTE~, V. (1966). Synchronization of mitosis by the fusion of the plasmodia of Physarum polycephalum. J. Cell Biol. 31, 204-209. SACHSENMAIER, W., and RUSCH, H. P. (1964). The effect of 5-fluoro-2’-deoxyuridine on synchronous mitosis in Physarum polycephalum. Exp. Cell Res. 36, 124-133. SACHSENMAIER, W., DONGES, K. H., RUPFF, H., and CZIHAK, G. (1970). Advanced initiation of synchronous mitoses in Physarum polycephalum following UV-irradiation. 2. Naturforsch. B 25,866-871. SUSSMAN, M., and SUSSMAN, R. R. (1962). Ploidal inheritance in Dictyostelium discoideum: stable haploid, stable diploid and metastable strains. J. Gen. Microbial. 28, 417-429. VON STOSCH, H. A. VAN ZUL-PISCHINGER, M., and DERSCH, G. (1964). Nuclear phase alternance in the myxomycete Physarum polycephalum. Abstr. 10th Int. Bot. Congr., pp. 481-482. WHEALS, A. E. (1970). A homothallic strain of the myxomycete Physarum polycephalum. Genetics 66, 623-633. YEMMA, J. J., and THERRIEN, C. D. (1972). Quantitative microspectrophotometry of nuclear DNA in selfing strains of the myxomycete, Didymium iridis. Amer. J. Bot. 59, 828-835. YOUNG, H. D. (1962). “Statistical Treatment of Experimental Data” pp. 101-126. McGraw-Hill, New York.