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Genetics Department, Ressarch School of Biological Sciences, The Australian National University, P.O. Box 475,

Canberra City, A.C.T. 2601, Australia Manuscript received December 12, 1977

Revised copy received April 11, 1978


Strains of Dictyostelium discoideum resistant to cobaltous chloride have been isolated at a frequency of approximately 10-6. The resistant strains have one of three phenotypes, recessive to wild type, dominant to wild type and dominant to wild type hut requiring the presence of cobaltous chloride to maintain resistance. Strains carrying a dominant cobaltous chloride resistance mutation and a recessive growth temperature-sensitive mutation can be mixed with wild-type haploid lines and then subjected to selection so that only diploid lines survive. Differential sensitivity to cycloheximide has also been observed. Hypersensitivity to cycloheximide in combination with dominant cobaltous chloride resistance provides a means of selecting diploids without the use of temperature-sensitive mutations.


present, the parasexual cycle is the only practical vehicle for genetic analysis in the cellular slime mold, Dictyostelium discoideun. The first step in the cycle involves the formation of a diploid strain by the fusion of t w o haploid strains. Although diploids have been isolated without using a selective system (SUSSMAN and SUSSMAN 1963; LOOMIS an8 ASHWORTH 1968), in general such methods are unsatisfactory because diploid formation is rare (-le5). Presently, there are three practical selective systems, all of which are based on the use of recessive mutations involving growth temperature sensitivity (LOOMIS 1969; KATZ and SUSSMAN 1972), ultraviolet light or y-ray sensitivity (WELKER and DEERING 1976), or sensitivity to Bacillus subtilis, cycloheximide or methanol (NEWELL et al. 1977). All of these systems have the disadvantage that for diploid isolation both haploid strains must carry one of the above mutations, so that direct selection can be made for complementing diploids by the removal of haploids. It would be desirable to devise a system such that only one haploid has to be genetically marked. A growth temperature-sensitive haploid strain carrying a dominant resistance mutation could provide such a system. This would allow easy integration into the genetic system of strains that carry various kinds of muta- tions, e.g., developmental mutations, but do not carry mutations which can be selected against (e.g., growth temperature sensitivity, etc.). This report describes



the isolation of mutants exhibiting dominant resistance to cobaltous chloride and demonstrates their application in parasexual genetic analysis.


Media: SM-agar ( SUSSMAN 1966) and inhibitor-containing SM-agar were prepared exactly as described previously, except that Calbiochem agar (11 g/l) replaced Oxoid agar No. 3 (WILLIAMS and NEWELL 1976). An appropriate amount of cobaltous chloride was added to the melted agar from a stock solution of 100 mg/ml CoCl2.6H,O (May and Baker, Ltd., Dagenham, England).

Strains: D. discoideum was grown on a cobalt resistant strain of Klebsiella uerogenes at 21 zk 1" (WILLIAMS and NEWELL 1976). D. discoideum strains used are all derivatives of the NC4 isolate and are described in Tables 1 and 2. Methods used to maintain stocks have been described previously (WILLIAMS and NEWELL 1976).

Tests for recessiveness or dominance: Diploids were constructed using techniques described previously (WILLIAMS and NEWELL 1976). In brief, amoebae of two strains to be crossed were scraped with toothpicks from clonal plates and swirled together in 0.1 ml of 520 mM CaC1, i n a well of a Falcon Microtest I1 tissue culture plate (Cat. No. 3040) with lid (Cat. No. 3041). After shaking for 17 hr at 22", the entire contents of a well were transferred to a nutrient agar plate. In most cases the diploids were isolated on SM agar a t 26.8" +- 0.2", although in some experiments involving dominant cobalt resistant strains, diploids were isolated on SM-CoC1,.6H2O (300 o r 350 cg/ml) agar at 26.8" 5 O.L?', or at 21" +. 1" on SM agar containing CoC1,.6HZO (200 pg/ml) plus cycloheximide (100 pg/ml). Dominance or recessiveness of cobalt resistance mutations (cob) was tested by plating amoebae of diploids heterozygous for a cob mutation clonally on SM-agar and SM-agar containing cobaltous chloride. Haploid segregants were obtained by plating diploids that were heterozygous for an mrA mutation on 2% methanol -SM agar, as described previously (WILLIAMS, KESSIN and NEWELL 1974). Haploids were obtained from other diploids by plating diploid amoebae clonally on SM-agar containing 20 pg/ml or 50 pg/ml of the haploidizing agent ben late (WILLIAMS and BARRAND 1978).


Isolation of mutants resistant to cobaltous chloride: Wild-type strains are sensi- tive to CoCl, * 6H2O at levels above IQ0 pg/ml. Table 3 shows that in strain "81




Plating efficiency of cobaltous chloride resistant mutants

Strain Parent

NP62S X2 HU24ll X22 HU26 x 2 2

HU32 NP81

HU344J NP81 HU357 NP81 HU367 NP81

HU50 NP170

HU5l** NP170 HU52** NP170 HU54** NP170

% Growth rate* % Plating efficiency$

cob efficiency on (ag/ml CoCl,.GH,O)

% Plating O n CoC1,.6H,O -I- SM compared to SM alone

mutation SM agar 300 400 500

A1 -35 1 -353 -354 -355 -356 -357 -359 -360 -361 -363 100 100 100 80 50 70 80 50 15 20 35 -20(300) -10(300) -10(300) -80(300.)


1 O(3OO) <10(300)


10 (300) -70(4QO) -50(400) -20(400) -508(400)

1 W 2 ) 9 4 t 6 (4) 97 t 6 (4) 68 t 10( 11) 4 8 + 1 9 ( 3 ) t 38 (2)


52.3 (2)


N T N T 1 W l ) t NT

NT: Not tested.

All mutants are of certain independent origin.

Amoebae from a single clone on SM agar were plated clonally on SM agar and SM agar


CoCl,~GH,O except for HU32, in which case amoebae were taken from SM agar


300 pg/ml CoCl,~GH,O rather than SM agar.

* Measured as colony diameter after seven o r 14 days, numbers in brackets refer to the level of


Slow growth, colonies take seven to 14 days to appear. CoC12.6H,0.

Numbers in brackets refer to number of experiments. Standard errors were calculated where


three o r more experiments were performed.


Identical to strain HU26 (Table l ) , except for a different cob mutation as shown here.

7 Identical to strain HU32 (Table l ) , except for a different cob mutation as s h m here. ** Identical to strain HU50 (Table l ) , except for a different cob mutation as shown here.

in a ring 1 cm from the edge of the cobaltous chloride SM-agar plate. More details of mapping with cobalt mutants, which is less straightforward than with, for example cycloheximide-resistant mutants, will be described elsewhere (WILLIAMS, in preparation). I n plates containing extremely high levels of CoCI,


6H,O (e.g., 500 pg/ml), there is often a pinkish precipitate a t the center of the plate. This area is relatively nontoxic, presumably because the cobalt has become complexed in some way. This is not a problem, as most studies are done with plates containing 350 pg/ml o r 400 pg/ml CoCl, .6H,O, which do not have such precipitates.


CoC12 RESISTANCE IN D. discoideum


Sensitivity of wild-type strains of D. discoideum to CoC1,.6H20 and frequency of isolation of resistant mutants


Frequency of spontaneously occurring resistance to cobaltous chloride$ Level of CoC1,.6H20 (pg/ml)

Strain Toxic level of CoCI,.6H,O 300 400 500

NP81 90% PE* at lOOpg/ml; <1%

P E at 200 lg/ml 7.7 i 3.3


10-7 N T t N T t Some leakine, and poorly

resistant colonies at a frequency

of 7.9 k 2.5


1W at 3001 pg/ml Slight leakiness at 300 fig/ml after prolonged incubation of

106 amoebae/plate 1.6 X 10-6 NTf NTf.

Serious leakiness at 300 pg/ml after prolonged incubation of

106 amoebae/plate 6.4 2 2.1


10-7 9.1 x 10-8 1.8 x 10-711



less thans

* PE, plating efficiency.


NT, not tested.

Frequency k standard error based experiment involving screening of 2.3


$j No mutants o r leakiness observed in


Three mutants obtained from 1.7 x

over a pink precipitate and thus were pg/ml (see text).

on four experiments, except for X22 in which only one

107 amoebae has been conducted. 1.1 x 107 amoebae plated.

: 1 0 7 amoebae plated, but all grew in the center of plates exposed to an effectively laver concentration than 500

on linkage group VII, strains HU50, HU51, HU52 and HU54 were constructed to have tsgG and cob mutations in cis for l i d a g e studies. These strains often fruited better on SM-agar containing cobaltous chloride than on SM-agar alone. Most of the mutants shown in Table 2 grew on SM-agar containing cobaltous chloride at less than 20% the rate on SM agar. Thus, in a number of cases colonies did not appear on CoC1, SM-agar until between seven and 14 days, com- pared to three to six days on SM-agar. Only strains HU32 (but see Table 5), HU50, HU51 and HU54 grew on CoCI, SM-agar at rates approaching their growth rate on SM agar. All the strains tested showed a n appreciable plating efficiency on SM agar containing 400 pg/ml CoC1,. 6H,O. To avoid problems of leakiness, routine studies with most of the mutants in Table 2 are now conducted using either 350 pg/ml or 400 pg/ml CoC1,. 6H,O.




Dominance of cob mutations in D. discoidem

Dominance/recessiveness* Level of CoCI,~GH,O (ag/ml)

Cobaltous chloride

Diploid Haploid parentsf resistancemutations 300 350 400 500

DU69 HU20/NP62


cobAI R R

DUI 02 NP81/HU24


cob-351 R R

DUI33 NP81/HU26


cob-353 D D (D) (D)

DUI71 NP 1 70/HU26


cob-353 D D (RI (RI

DU98 X22/HU32


cob-354 D D D D

DUI32 XS?2/HU36


cob-357 (D) (D)

DUI72 X22/HU50


cob-359 R R R (RI

DUI 74 M28/HU5 1


cob-360 R R R (RI

DU198 X57/€IU52


cob-361 R R R

DUI 68 X22/HU54


cob-363 R R R (RI

Diploid amoebae from SM-agar plates were used in all cases, except for DU98 in which amoebae from SM-agar


CoCl,~GH,O (300 pg/ml) were used.

* D: dominant to wild-type; (D) : dominant to wild-type but with low plating efficiency or very slow growth rate on SM-agar


CoC1,6H,O; R: recessive to wild-type; (R) : recessive to wild- type but haploid segregants barely grow on SM-agar


CoC12.6H,0. A blank space shows that no colonies were obtained on these plates; the cobalt resistance mutations in the original haploids were essentially sensitive to this level of CoCl,~GH,O.

f Parents are described in Tables 1 and 2 except for: HU20, a cycloheximide resistant (cyc-352: slow growth on 400 pg/ml) mutant of X22 which was isolated in this laboratory; M28, K A n and

SUSSMAN (1972); X57 a Bacillus subtilis sensitive strain derived from DP746 (X22/NP194) NEWELL et al. (1977).

was not examined in this way because of the complexity due to loss


cobalt resistance in strains carrying cob-354 when passaged in the absence of CoCl,

(see below). However, DU98 was shown to be heterozygous for other chromo- somal markers (e.g., bwnA/+, whiA/+, ebrA/+, acrA/+) and hence is almost certainly heterozygous at the cob-354 locus.




discoideum 43 efficiency in a heterozygous diploid even at 500 pg/ml CoCl, 6H,O. However, unlike the other


mutations discussed here, resistance to cobaltous chloride in strain HU32 is lost by passage under nonselective conditions. Cobaltous chloride resistance can be maintained when H u 3 2 is passaged on levels of CoC1,. 6H,O that are not toxic to wild-type strains. There is some variation in W e r e n t experi- ments, but after one passage on 100 pg/ml CoCl2.6H,O, which allows 90% plating efficiency of wild-type cobalt-sensitive strains, the plating efficiency when retested on 300 pg/ml CoCl, 6H,O was between 10% and 40%. Two such experi- ments are shown in Table 5. After two passagings on 100 pg/ml CoC1,. 6H,O, the plating efficiency of HU32, when retested cm 300 pg/ml CoCl, .6H,O, was still between 1 and 5



This shows that HU32 can be passaged at least twice on

100 pg/ml CoCl, . 6H20 without resistance to 300 pg/ml CoCl,. 6H,O being lost. The nature of the cob-354 mutation has not yet been examined further.


test whether the dominant CoCl, resistance mutations in strains HU26 and HU32 would be useful for parasexual genetic analysis, these strains were crossed to the wild type (not growth temperature sensitive and CoC1, sensitive) strain AX3, and diploids were selected at the restrictive temperature (to kill HU26 or HU32) on SM


cobaltous chloride 300 pg/ml (to kill AX3).



cases diploids have been isolated at a frequency of about even using the simplified technique of




(1976). Strain AX3 shows some leakiness on 300 pg/ml CoC12*6H20, but the diploids were picked off as discrete tiny colonies after four days at 26.8" before leakiness became serious. They were puri- fied by clonal passage and shown to be heterozygous f o r the appropriate markers, including the cob mutations (Table 1) by segregating haploids on 2% methanol SM-agar (AX3 X HU26) or 30 pg/ml ethidium bromide SM-agar (AX3 X



Plating efficiency of strain HU32 on SM-agar containing various levels of cobaltous chloride

Previous growth conditions.

% Plating efficiency on SM 4- 300 pg/ml CoCI,.GH,O compared to that on SM alone

Expt. 1 Expt. 2












< I < I

11 43 61








* Amoebae from a single clone an SM agar containing 300 pg/ml C&1,6H20 were plated clonally on the media shown. When colonies were approximately 1 cm diameter, amoebae from each treatment were plated clonally on SM-agar and SM-agar


300 g/ml CoCl,.GH,O.


44 K. L. W I L LI A M S

Isolation of diploids using a combination of drug resistance and drug sensi- tivity: In the course of studies on the levels of resistance of several parent strains to various inhibitors, a pronounced difference was noticed in the level of sensi- tivity to cycloheximide in “sensitive” strains. Strain HU26 had a plating efficiency of about 1% on 100 p g / d cycloheximide and grew slowly. On 200 pg/ml cycloheximide and above, the plating efficiency was less than lo+. Several axenic strains derived from strain AX3 plated with close to 100% efficiency on 100 pg/ml cycloheximide and showed appreciable plating efficiency on 200 pg/ml cycloheximide. Thus, HU26 was characterized as exhibiting heightened sensi- tivity to cycloheximide, which could conceivably be recessive to wild-type sensitivity (of axenic strains). The possibility of selecting against HU26 on the basis of its hypersensitivity to cycloheximide instead of its temperature sensitivity was investigated. Table 6 shows the results of plating HU26, NP81 and DU133 on various combinations of cycloheximide and cobaltous chloride. It will be seen that the combination of cycloheximide 100 p g / d and cobaltous chloride 200 pg/ml results in a plating efficiency of less than


X of each haploid, but allows about 50% plating efficiency of DU133. Diploids were selected at a fre- quency of about 3 x 1 0-6 from the fusion of HU26 and NP81, using coaggregation of amoebae in 20 mM CaC1,




1976) and plating at 21”


Establishment of appropriate levels of cobaltous chloride and cycloheximide to allow isolation of

diploids on S M agar plates containing a combination of these inhibitors at 21”

SM agar containing Time (days CoCI,~6H20/cycloheximide (pg/ml)t Strain Amoebae/plate* at 21’) 0/0 300/50 300/100 300/200 200/100 200/200

HU26$ 2.7


105 7 G SG NG NG NG NG

(haploid) 14 G G NG NG SG NG

NP81S 2.5 x 1015 7 G NG NG NG NG NG

(haploid) 14 G NG NG NG NG NG

DUI33 22 or 110 7 1 0 20 3 0 51 13

(Diploid (30) ( < I ) ( < I ) (1-12) (1-2)

HU26/NP81) 14 100 30 3 0 54(1 14

(1-20) (1-3) (20) (6-8) Plating efIiciencyll

* Numbers based on haemacytometer counts. For the diploid DU133, 22 amoebae were plated

on the control (no inhibitor) plate, while 110 amoebae were added to the inhibitor-containing plates.

f. G: growth and fruiting; NG: no growth; SG: m e signs of growth, often on small areas of

the plates.

$ HU26 is resistant to CoC1,.6H,O (dominant) and extra-sensitive to cycloheximide (recessive).

S NP81 is wild type for both CoCl,.6H2O and cycloheximide sensitivity.


Plating efficiency, expressed as a percentage of the control plates (O/O, no inhibitor). Numbers




discoideum 45 on SM-agar containing 100 pg/ml cycloheximide and 200 pg/ml cobaltous chloride.


This report describes the characteristics of dominant resistance mutants in Dictyostelium discoideum. These mutants will allow the incorporation of strains carrying various kinds of mutations into the genetic system. For example, hetero- zygous diploids were constructed from a haploid strain containing no markers that could be selected against (AX3) and a haploid strain carrying a dominant cobalt resistance mutation and a growth temperature sensitivity mutation (HU26 or HU32). This was done by plating at the restrictive temperature (killing HU26 or HU32) on agar plates containing cobaltous chloride (killing AX3). Alterna- tively, a diploid was isolated between haploid strains AX3 and HU26 by plating on SM-agar containing cycloheximide and cobaltous chloride at 21 O , thereby

exploiting the dominant resistance to cobaltous chloride and the naturally occurring recessive hypersensitivity to cycloheximide of strain HU26. Mutants exhibiting recessive hypersensitivity to both cycloheximide and methanol have been isolated in strain AX3 previously by NEWELL et al. (1977), although those workers found that their mutants were unsatisfactory for general use as they grew poorly and were developmentally aberrant even on SM-agar.

All of the recognized resistance mutants previously described, which have been characterized genetically, carry mutations that are recessive to wild type. These are mutations at cycA (SINHA and ASHWORTH 1969; KATZ and SUSSMAN 1972;





and KAO 1974; ROTHMAN and ALEXANDER 1975; COUKELL 1975 ; WILLIAMS 1976), acrB (WILLIAMS, KESSIN and NEWELL 1974), a c e (ROTHMAN and ALEXANDER 1975) and ebrA



and NEWELL 1977). Mutations so far isolated at two new resistance loci, which possibly map on linkage group I (cadA, resistance to cadmium chloride and cyhA, resistance to cyclohexanol), are also recessive to wild type (WILLIAMS and NEWELL, unpublished).


and TAKEUCHI (1971) and FUKUI and MIYAKE (1975) have used mutants resistant to naromycin (cyclo- heximide) and trichomycin for genetic analysis in


discoideum. It is doubtful that diploids were isolated in these studies, but double-resistant strains were obtained. The dominance or recessiveness of the resistance mutations is yet to be clearly established, although it seems likely that resistance to at least one, if not both, of these inhibitors is recessive to wild type.


46 K. L. W I LL I A M S

suppresses brown pigment production is currently being investigated. Secondly, the cobalt resistance mutations are of interest because it is likely that they form at least one complementation group that marks linkage group







unpublished) ; for


reason detailed mapping studies are in progress on all of the cob mutations isolated in this study. I thank GILL ROBSON for technical assistance and E. SMITH and B. ROLPE and the referees for helpful criticisms.


COUKELL, M. B., 1975

FUKUI, Y. and Y. MIYAKE, 1975

Parasexual genetic analysis of aggregation-deficient mutants of Dictyo stelium discoideum. Molec. Gen. Genet. 142: 119-135.

Parasexual hybridization in cellular slime molds. I. Appear- ance of hybrid clones at high frequency in a short period and its relation to cell fusion in Dictyostelium discoideum. Cell Struct. Funct. 1 : 23-31.

FUKUI, Y. and I. TAKEUCHI, 1971 Drug resistant mutants and appearance of heterozygotes in the cellular slime mould Dictyostelium discoideum. J. Gen. Microbiol. 67: 307-31 7. KATZ, E. R. and V. KAO, 1974 Evidence for mitotic recombination in the cellular slime mould

Dictyostelium discoideum. Proc. Natl. Acad, Sci. U.S. 71 : 4025-4026.

KATZ, E. R. and M. SUSSMAN, 1972 Parasexual recombination in Dictyostelium discoideum: Selection of stable diploid heterozygotes and stable haploid segregants. Proc. Natl. Acad. Sci. U.S. 69: 495-498.

LOOMIS, W. F., 1969 Temperature-sensitive mutants of Dictyostelium discoideum. J. Bacterid. 99: 65-69.

LOOMIS, W. F. and J. M. ASHWORTH, 1968 Plaque-size mutants of the cellular slime mould Dictyostelium discoideum. J. Gen. Microbiol. 53: 181-186.

NEWELL, P. C., R. F. HENDERSON, D. MOSSES and D. I. RATNER, 1977 Sensitivity to Bacillus subtilis: A novel system for selection of heterozygous diploids of Dictyostelium discoideum. J. Gen. Microbiol. 100 : 207-21 1.

ROTHMAN, F. G. and E. T. ALEXANDER, 1975 Parasexual genetic analysis of the cellular slime mold Dictyostelium discoideum A3. Genetics 80: 715-731.

SINHA, U. and J. M. ASHWORTH, 1969 Evidence for the existence of elements of a parasexual cycle i n the cellular slime mould Dictyostelium discoideum. Proc. Roy. SOC. B 173: 531-540.

SUSSMAN, M., 1966 Biochemical and genetic methods in the study of cellular slime mold devel- opment. pp. 397409. In: Methods in Cell Physiology, Vol. 2. Edited by D. PRESCOTT. Academic Press, New York.

PIoidal inheritance in the slime mould Dictyosrelium discoideum: haploidization and genetic segregation of diploid strains. J. Gen. Microbiol. 30: 349-355.

WELKER, D. L. and R. A. DEERING, 1976 Genetic analysis of radiation-sensitive mutations in

WILLIAMS, K. L., 1976 Mutation frequency at a recessive locus in haploid and diploid strains

WILLIAMS, K. L. and P. BARRAND, 1978 Parasexual genetics in the cellular slime mould Dicfyostelium discoideum: haploidisation of diploid strains using ben late. FEMS Microbiol. Letters (in press).

SUSSMAN, R. R. and M. SUSSMAN, 1963

the slime mould Dictyostelium discoideum. J. Gen. Microbiol. 97: 1-10.







WILLIAMS, K. L., R. H. KESSIN and P. C. NEWELL, 1974 Parasexual genetics in Dictyostelium discoideum: mitotic analysis of acriflavin resistance and growth in axenic medium. J. Gen. Microbiol. 84: 59-69.

WILLIAMS, K. L. and P. C. NEWELL, 1976 A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis. Genetics 82 : 287-307.

WRIGHT, M. D., K. L. WILLIAMS and P. C. NEWELL, 1977 Ethidium bromide resistance, a selective marker located on linkage group IV of Dictyostelium discoideum. J. Gen. Microbiol

1012: 423426.




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