A ALCA, THE FORM EXPRESSED IN PHYSARUM POLYCEPHALUM · A GENE, ALCA, AFFECTING THE LIFE CYCLE FORM EXPRESSED IN PHYSARUM POLYCEPHALUM CHRISTINE L. TRUITT1, CHARLES S. HOFFMAN2 AND

A GENE, ALCA, AFFECTING THE LIFE CYCLE FORM EXPRESSED IN PHYSARUM POLYCEPHALUM

CHRISTINE L. TRUITT1, CHARLES S. HOFFMAN2 AND CHARLES E. HOLT

Departmeni of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Manuscript received February 16, 1981 Revised copy accepted March 2,1982

ABSTRACT

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called “amoebaless life cycle” or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, &A. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spare germination, but also affecting the phenotype of alc amoebae, which occasionally arise from d c spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) f o r m plasmodia by the sexual mechanism less efficiently than wild-type amoebae. The various effects of an aZc mutation seem to derive from mutation of a single gene, since reversion for one effect is always accompanied by reversion for the other effects. Moreover, a mutation, aptAl, that blocks direct plasmodium formation by aZcA amoebae, also increases their growth rate to near normal. The manner of plasmodium formation in aZcA strains differs significantly from that in another class of mutants, the gad mutants. Unlike gad amoebae, alcA amoebae need not reach a critical density in order to differentiate directly into plasmodia and do not respond to the extracellular inducer of differentiation. In addition, &A differentiation is not prevented by a mutation, npjAl, that blocks direct differentiation by most gad amoebae.

HE Myxomycete Physarum polycephnlum has a sexual life cycle that in- cludes two distinct vegetative forms: the microscopic, uninucleated amoebal

form and the macroscopic, multinucleated plasmodial form (DOVE and RUSCH 1980). Under appropriate conditions, two compatible haploid amoebae “mate,” i.e., form a zygote that differentiates into a diploid plasmodium. Under starva- tion conditions in the presence of light, the plasmodium produces fruiting bodies containing haploid spores derived from meiosis. Germination of the spores

Present address Department of hIicroblology, The University of Texas Health Science Center at San Antomo, Texas 78281.

2 Present address Dcpartmcnt of Molecular Biology and Msrobiology, Tufts University School of Medicine, Boston, hhssachusetts 0211 1.

Genctics 101 : 35-55 Mdy, 1082

36 C. L. TRUITT, C. S. HOFFMAN A N D C. E. HOLT

results in the emergence of haploid amoebae, thus completing the sexual life cycle. An asexual cycle is also known in which individual haploid amoebae “self,” i.e., convert directly into haploid plasmodia (ANDERSON, COOKE and DEE 1976). Such plasmodia are capable of fruiting and producing a few viable haploid spores (LAFFLER and DOVE 1977). Amoebae obtained from natural isolates form plasmodia readily only by mating. Such amoebae, which are referred to as “nonselfing” or “heterothallic” amoebae, will in fact self, but only at an exceedingly low frequency (ADLER and HOLT 1977). Mutant amoebae that self readily can be obtained and are referred to as “selfing” or “apogamic” amoebae. These amoebae can also mate, and under appropriate conditions many do SO

re ad i 1 y . The striking metamorphosis of amoeba to plasmodium has been the subject

of a number of studies designed to elucidate the physiological and genetic factors initiating differentiation ( GORMAN and WILKINS 1980). A multiallelic mating specificity gene, matB, has been shown to regulate cell fusion in the sexual cycle; only amoebae carrying different matB alleles fuse readily (YOUNG- MAN et al. 1979; YOUNGMAN, ANDERSON and HOLT 1981; HOLT et al. 1979). Another multiallelic gene. matA (also called mt) , regulates the fate of diploid zygotes, in that only those containing two different alleles of matA differentiate to the plasmodial form (DEE 1966; ADLER and HOLT 1975; YOUNGMAN, ANDER- SON and HOLT 1981). Certain of the mutations that enhance selfing ability are referred to as gad (greater asexual differentiation) mutations. Nearly all of these are tightly linked to matA (ADLER and HOLT 1977; SHINNICK and HOLT 1977; GORMAN. DOVE and SHAIBE 1979; SHINNICK, ANDERSON and HOLT, in preparation). Also known are mutations that either block or modulate mating and selfing (WHEALS 1973; ANDERSON and DEE 1977; SHINNICK et al. 1978; YOUNGMAN, et al., in preparation). In the present work, we describe the analysis of a gene, &A, which recombines freely with matA and matB and controls, in a manner qualitatively different from the other genes, whether the amoeba1 o r plasmodial form is expressed.

The amoebaless life cycle or alc mutants were first recognized as a subclass of rare plasmodia isolated from heterothallic amoebae ( ADLER, DAVIDOW and HOLT 1975). The majority of these plasmodia were actually nonmutant, haploid selfs. The remaining plasmodia were classified about equally as alc mutants, whose spores give rise to haploid plasmodia directly and gad mutants, whose spores give rise to selfing amoebae (ADLER and HOLT 1977). One of the alc mutants was shown to contain a single Mendelian allele (designated alc-1) responsible fo r its phenotype. (It was possible to analyze this mutant because some of its spores germinated as amoebae; these amoebae still carried the alc mutation and would mate with wild-type amoebae.)

The alc-l mutation, besides affecting the type of cell which emerged from a spore, also coderred a selfing tendency on those amoebae that did emerge. The mutation was thus expressed in two rather different physiological states, en- couraging the view that the alc gene might play a direct role in a control mechanism determining the differentiated form adopted by the organism. But it was

Physarum alcA MUTANTS 37

also conceivable that the trlc mutations were complex, that their different effects on the re-establishment of the amoebal form in a germinating spore and the maintenance of the amoebal form during growth were caused by alterations in different linked genes. Even if a2c-1 represented a mutation in a single gene, there was another disquieting possibility suggested by the observation that alc-2 amoebae grew rather slowly, particularly under the condition (30" ) at which they selfed most frequently. This suggested that each alc mutant might contain a mutation in one of a relatively large set of genes of particular importance to amoebal growth, and that the relative preference of alc cells for the plasmodial form might be only an indirect consequence of growth mutations. We present evidence that the pleiotropic phenotype of three alc mutations arises from mutation of a single gene, designated &A, which is likely to play a central role in determining which life cycle form is expressed.

MATERIALS AND METHODS

Strains: All amoebal strains had the Colonia genetic background (ADLER and HOLT 1974; COOKE and DEE 1975). The alc mutant strains analyzed were CH918 (carries &A8 and was derived from strain LU911), CH967 (elcAiU, LU896), CH970 (aZcAII, CH351) and CH989 (alcA9, LU896). Other strains are listed in Table 1.

Culture conditions: Routine culture procedures were conducted as described by ANDERSON (1979), except that PYE medium was sometimes used instead by PRM medium, and bacterial suspensions were obtained by washing off a nutrient agar plate of E . coli in either 100 ml or, when a concentrated suspension is specified, 10 ml sterile H,O. Peptone yeast extract (PYE) is essentially as devised by E. N. BREWER and A. PRIOR and contains, per liter, 10 g N-Z Case (a casein hydrolysate, 3 g yeast extract, 9 g glucose, 4.2 g citric acid.H,O and 0.5 g MgSO4-7H,O. The medium is brought to pH 4.6 with 30% KOH, and IO ml of 0.5% hematin is added after sterilization. Amoeba1 strains were grown at 26" unless otherwise indicated, except that nlc strains were routinely grown at 21" to inhibit differentiation to plasmodia. Cultures of microplasmodia were begun by transferring sections of plasmodia grown on PYE-agar (equal volumes of PYE and 3% agar) to a 250 ml flask containing 50 ml PYE and shaking vigorously at 26" for several days before dilution to fresh liquid media. For the measurement of dry weight, microplasmodia were collected by centrifugation, washed with H,O to remove slime, and dried to constant weight under vacuum at 90".

Mutant hunt: Rare plasmodia were isolated from spot cultures of nonmutagenized amoebae on dPRM agar (ADLER and HOLT 1977). Most cultures were incubated at 30", as ADLER and HOLT (1977) found that a lower percentage of plasmodia isolated at this temperature were nonmutant than at lower temperatures. Fifteen inbred strains (Table I) were used as parents in the mutant isolation. A spot culture was prepared by inoculating 0.1 ml of a bacterial suspension containing amoebae at I O 5 cells/ml onto the center of a week-old agar plate and allowing the fluid to absorb without spreading. Plasmodia rising from spots were cut out, further cultured on PRM-agar, and induced to sporulate

Of a total of 2688 spot cultures set up, 53 yielded plasmodia (Table 2), generally after two to three weeks incubation. Spores from the plasmodia were germinated, and the progeny were examined. Plasmodia were given the following designations: alc mutants, when some or all of their spores germinated as plasmodia; gad mutants, when all their spores germinated as amoebae that subsequently selfed; and nonmutant, when spores gave rise t u nonselfing amoebae only (Table 2).

The 31 plasmodia designated aZc mutants arose from eight amoebal strains (CH326, CH351, CH354, CH395, LU912, LU688, LU896, LU911) representing each of the four mutA types. In several cases, more than one alc plasmodium arose from the replicate spot cultures set up from

38 C . L. TRUITT, C. S. HOFFMAN A N D C. E. HOLT

TABLE 1

Amoebal strains

Strain niat.4-i mntB fiis.4

CH326* CH351* cH354' CH395 * CH508' CH768 * CH801* CH814 CH818 CH819 CH836 CH921 CH929 CH99O CH991 LU648* LU688' LU865 LU869 LU887* LU896* LU897* LU898* LU911* LU912'

4 3 4 4 2 4 2 3 ht ht 3 3 I 3 3 I 2 1 I 2 I I 2 3 3

I I 3 2 3 2 2 3 2 2 3 I 3 I 3 I I I I I I I I .3 I

2 2 I 2 2 2 I I I 2 I I I 2 2 I I I I I 2 2 2 2 2

U hi Other

4- fusC2 + + + + + npfA1 3 + + gad-h aptAI + gad-h aptA1 3- gad-5 npfAI + gad-5 npfAI I + gad-5 npfAI t gad-5 npf.41 + -c + aptAI + npfAI I

L

All the above strains except CH326 carry the fusCI allele. * Strains used in mutant hunt; see MATERIALS AND METHODS. + The allele matAh (or mt-h) may be regarded as matA2 gad-h, where gad-h is a mutation

in or close to matA2 (ANDERSON 1979; ANDERSON and HOLT 1981).

TABLE 2

Analysis of plasmodia from mutant hunt

Number of cultures screened or plasmodia found 21" 26" 30" Total

Amoebal cultures screened 160 480 2048 2688 alc plasmodia 3 2 26 31* gad plasmodia 2 2 15 19 Nonmutant plasmodia 0 1 2 3

* Of the 31 alc plasmodia, 11 were judged to be independent mutants; see MATERIALS AND METHODS.

a single amoeba1 suspension. Generally, these plasmodia were considered likely to contain the same mutation, and only one was analyzed further to ensure that the alc mutations studied were independent. In two cases, however, two alc isolates from the same set of replicate spot cultures had different phenotypes and were therefore considered likely to possess independent mutations and were retained for further study.

Physarum alcA MUTANTS 39 Differentiation tests: Amoeba1 strains were tested for selfing ability in spot cultures on

dPYE-agar plates incubated at the appropriate temperature (30" for determination of the presence of an alc mutation) and checked periodically for the presence of plasmodia.

Mating tests: The m t A and matB alleles of an amoebal progeny strain from a cross were determined by setting up a set of four spat cultures, each of which contained the strain to be tested and a tester amoebal strain. The spot cultures were prepared on non-nutrient agar plates (3 mM Na citrate, pH5, 10 mM MgSO,) with concentrated bacteria and equal numbers of the two strains. The tester strains comprised each of the four possible matA, matB combinations ob- tainable from the given cross. The cultures were incubated at 21" (to prevent alc amoeba1 differentiation) for about four days, a t which time plasmodia were visible in the one culture in which the tester possessed different matA and matB alleles than the strain being analyzed. Determina- tion of plasmodial markers ( f u d , whi) could then be determined as described elsewhere (ANDERSON and DEE 1977).

Crosses involving alc amoebal strains: Attempts to mate alc amoebae with alc+ amoebae or other alc amoebae were conducted at 21" to suppress selfing and involved strains differing at matA, m t B , fusA and sometimes whi. The latter two are plasmodial markers that permitted us to determine whether plasmodia from mating mixtures arose by selfing. Plasmodia appeared later in some attempted alc+ x alc mixtures than in comparable a b + x alc+ mixtures and were not always the result of mating, particularly with alc+ x aZc-9. Even the plasmodia that arose relatively early in mating mixtures involving alc amoebae sometimes possessed the same plasmodial markers as one of the parent amoebal strains. (Such an apparent stimulation of selfing in mixtures of amoebae differing at matA has also been seen in alc+ strains; the stimulation was detectable only under suboptimal mating conditions (ANDERSON 1976; ANDER- SON and TRUITT, in preparation)). Some plasmodia arising from mating mixtures of alc x alc+ or alc x alc amoebae did exhibit the hybrid fusion type (fusAI/AP) expected of diploid plasmodia arising from the fusion of two amoebal strains. In several cases involving alc X alc mixtures, more rarely in alc+ x alc mixtures, the progeny derived from such a hybrid plasmodium contained only parental combinations of markers, indicating that nuclear fusion had not occurred following amoebal cell fusion.

Determination of amoebal growth and differentiation: The growth rates of alc or a b + amoebae and production of plasmodia at various temperatures were determined as described by YOUNGM~N et al. (1977), except that assay plates were incubated at 21' to prevent further a k differentiation after the samples were taken.

npfA analysis: The matA3 progeny of the cross CH918 matA3 matB3 fusA2 npfA+ alcA8 X LU869 matAI matBi fusAI npfAi' &A+ were analyzed for the npfA allele present by a complementation test developed by ANDERSON (1979). First, the progeny were analyzed for their matA, matB and fusA alleles and their selfing phenotype. Then they were mixed as described for mating tests above with a gad-5 npfAl strain carrying the same matA allele (SO that plasmodium formation based on different matA alleles would not occur), but different alleles of matB and fusA. The four gad-5 npfAl strains used were CH921, CH990, CH836, CH991 (see Table 1). The mixtures were incubated at 26", a t which temperature the gad-5 mutation is expressed. (Control spots of the alc strains were found not to produce visible plasmodia during the six-day incubation period.) The gad-5 mutation is dominant to gad+ in promoting the differentiation of an amoeba to a plasmodium; however, the recessive npfAI lesion suppresses gad-5-induced differentiation (SHINNICK 1978). Thus, if the progeny strain to be tested possessed the npfAl allele, the resulting diploid (gad+ npfAI/gad-5 npfAl) did not differentiate. If, however, the progeny strain possessed the npfA+ allele, the diploid amoebal cell (gad+ npfA+/gad-5 npfA1) did differentiate due to the dominance of the gad-5 mutation and the recessiveness of the npfAI mutation. Plasmodia arising from such mixtures were tested for hybrid fusion type ( fusAl / fusA2) to ensure that the plasmodia did not arise through selfing of either strain.

aptA analysis: Progeny of the cross CH918 matA3 matB3 fusA2 aptA+ alcA8 X LU865 matAI matBI fusA1 aptAI alcA+ were analyzed for the presence of the aptAI mutation by utilizing the fact that the recessive aptA1 lesion can prevent mating if present in both the amoebal strains mixed in a mating culture (WHEALS 1973). Each progeny strain was mixed in a spot

40 C. L. TRUITT, C . S. H O F F M A N AND C . E. HOLT

culture with an uptA2 strain (CH818 or CH819) that carried different alleles of matA and matB than either of the parental strains. Cultures were incubated at 21" to prevent selfing by &A progeny strains in the mixtures. After five days, the mating cultures were checked for the presence of plasmodia, which would indicate that the progeny strain carried an @A+ allele which allowed mating.

RESULTS

Isolation and preliminary characterization of new alc mutants: Most of the alc mutants isolated earlier (ADLER and HOLT 1977), including the one carrying alc-2, did not survive storage; for this reason, a new mutant hunt was conducted. A total of 2688 cultures of heterothallic amoebae were prepared, incubated and examined periodically for rare plasmodia. Of the 53 plasmodia found, 31 had the alc characteristic (Table 2), and these plasmodia appeared to be derived from 11 independent alc mutations (see MATERIALS AND METHODS).

Plasmodia carrying the 11 alc mutations were subjected to further analysis. Second generation plasmodia were obtained from individual spores of the original plasmodia and induced to sporulate. In each case, at least some of the second generation spores germinated as plasmodia, showing that the alc characteristic was heritable. The viability of the spores (usually 0.1 %-1%) was comparable to that of spores from gad or nonmutant haploid plasmodia.

Besides giving rise to plasmodia, the spores of 10 of the 11 mutants also gave rise to amoebae at frequencies ranging from IO-: to 5 x lo-' of the viable spores. (The remaining strain did not yield any amoebae upon germination of about lo3 viable spores.) The amoebae could, in principle, be original site revertants (i.e., alcf amoebae), suppressed mutants ( sup alc), or mutant amoebae (alc) that somehow escaped the effect of the mutation. One might expect that amoebae of the last type would tend to self because the mutants originated as rare plasmodia in cultures of heterothallic amoebae. Some or all of the amoebae from 9 of the 10 amoeba-yielding plasmodia did indeed self. Selfed plasmodia from these amoebae gave rise to spores that germinated as both amoebae and plasmodia, showing that the selfing amoebae still carried alc mutations. We searched unsuccessfully among clones of nonselfing amoebae from several of the mutants for a suppressed strain still carrying an alc mutation (data not shown). Spores of the tenth mutant gave only nonselfing amoebae, which were not analyzed further.

Crosses with alc+ amoebae: Attempts were made to mate alc amoebae from the nine mutants that gave rise to them with alc+ amoebae carrying different matA and matB alleles (see MATERIALS AND METHODS). The plasmodia that arose in the mating mixtures were often haploid aIc plasmodia rather than diploid plasmodia resulting from mating. The poor mating ability of alc amoebae is not due simply to their selfing tendency: some gad amoebae that self even more efficiently than alc amoebae mate readily (ADLER and HOLT 1977).

Successful alc x alc+ mating was obtained with three mutants, those carrying the mutations designated alc-8, alc-20 and alc-22 (Table 3) . Spores from the alc/alc+ plasmodia germinated as amoebae only, showing that the alc mutations


TABLE 3

Progeny analysis of alc x alc+ crosses*

CH918 matR4B3 alc-8 CH967 m a f A l B 1 alc-IO CII970 matA3Bf alc-I1 X X X

CfI801 matA2B2 ulc+ CII801 matAZB2 alcf CH%9 matAIB3 alc+ matA matR alc No. maul matB alc No. matA matB alc No.

2 2 2 2 2 3 2 3 3 2 3 2 3 3 3 3

+ 8 8 9

+ 8 8 6 f 5 8 4

+ 4 8 4

Total 48

1 1 + 9 1 1 10 2 1 2 f 8 1 2 10 3 Z I T I - 8 2 1 10 13 2 2 + 5 2 2 10 5

Total 53

1 1 1 1 1 3 1 3 3 1 3 1 3 3 3 3

+ 5 11 2 4 - 6 11 3 + 4 11 5 + 2 11 7 Total 34

* Amoeba1 progeny clones from the three crosses shown were analyzed for matA, matB and alc types. The alc genotype was based on the ability of amoebae to form plasmodia within seven (alc-8 and alc-11) or 10 (alc-10) days at 30". The contraction mat.43113 means matA3 matB3.

are recessive to alc+ in gemination behavior. Moreover, the results show that the alc+ product must be made before the final meiotic division and be able to act in an alc spore.

Progeny amoeba1 clones were established from the spores of the mated plasmodia and subjected to further analysis. Half the progeny of each of the alc-8/alc+, alc-lO/alc+ and alc-ll/alc+ plasmodia displayed the selfing tendency associated with alc amoebae (Table 3 ) . Selfed plasmodia from these progeny gave rise to spores that germinated as amoebae and plasmodia, con- firming the presence of the alc mutations. Thus it appears that a pair of alleles, alc and alc+, segregated in each cross, and that aEc causes both abnormal behavior of spores at germination and a selfing tendency of amoebae. The progeny analyses also showed independent segregation of matA, matB and alc (Table 3), as well as of whi, fusA and alc (data not shown), The alc-8 mutation was also shown to segregate independently of fusC (data not shown). Note that amoebae of different matA types displayed the selfing behavior associated with alc mutations. Thus, the alc mutations are expressed with different mating types. In additional experiments with alc-8, we showed that this mutation is expressed with matAl and matA4, as well as with matAB and mztA3 (Table 3 ) .

Crosses between alc mutants: To study linkage and complementation of the alc mutations, we attempted to mate amoebae carrying different alc mutations with one another. The alc-8, alc-ZO and alc-lI mutants were successfully mated in the three possible combinations. The spores from each of the alc/alc plasmodia germinated as a mixture of plasmodia and amoebae (Table 4) like the spores of the haploid mutants, but unlike the spores of alc/alc+ plasmodia. Thus, the three mutations lie in the same functional entity, which we designate alcA,

We also succeeded in mating alcA8 amoebae with alc-9 amoebae, even though alc-9 amoebae were never successfully mated with alc+. Spores of the alcA8 X

42 C. L. TRUITT, C. S. HOFFMAN A N D C . E. HOLT

TABLE 4

Amoeba1 plaques from alc spores

Genotype of p1n;modium Proportion of &ble

spores yielding amoeba1 plaque;

alc-8 alc-9 alc-10 alc-ll alc-8/alc-9 alc-S/alc-lO alc-S/alc-ll alc-I O/alc-ll

0.1-0.9 *

0.001 0.002 0.94 0.38 0.62 0.03-0.07

ca. 0.5-f

* Range for different experiments. -f The result for alc-9 is approximate, since spore behavior was not readily classified into two

categories. Some spores clearly gave rise to plasmodia directly, but others gave rise to small clones of amoebae that subsequently differentiated.

aZc-9 plasmodium also gave rise to plasmodia directly, so aZc-9 may also be in aZcA.

The proportion of aZc spores which germinated as amoebae rather than plasmodia varied considerably from mutant to mutant (Table 4). The proportion that germinated as amoebae was particularly high for aZcA8 spores, suggesting the possibility that residual aZcA+ activity is particularly high in the aZcA8 mutant. Heterozygous plasmodia carrying aZcA8 and each of the three other mutations also gave relatively high proportions of amoebal plaques (Table 4), as would be expected if the aZcA8 gene product were the most active of the mutant products. Although spores from &A10 or aZcAll haploid plasmodia gave 0.2% or fewer amoebal plaques, the aZcAlO/aZcAll diploid plasmodium gave about 5% amoebal plaques. One may regard this as partial complementation between the two mutants, although it may reflect a difference in spore formation in a haploid us. a diploid plasmodium.

Initial analyses of the progeny of the aZcA8/aZcAlO, aZcA8/aZcAll and aZcAlO/aZcAll plasmodia revealed no alc+ recombinants, showing that the three mutations are tightly linked to one another. Further progeny analyses demonstrated that the plasmodia were indeed diploid heterozygotes rather than haploid heterokaryons. Free recombination was observed between fusA and whi in the progeny of the aZcA8/alcAlO and aZcA8/aZcAlZ plasmodia (Table 5 ) and between matA and matB in the amoebal progeny of all three plasmodia (data not shown).

To search further for aZcA+ recombinants, additional amoebal progeny were analyzed for their selfing ability only. None of 184 amoebal clones (representing 300 total progeny) from an akA8 x aZcAll plasmodium failed to self. We did find three nonselfing (spot cultures, 21 days, 30") amoebal progeny among the spores of an aZcA8/aZcAlO plasmodium; approximately 1000 total progeny, including both amoebal and plasmodial plaques, were examined in the search for the nonselfing recombinants, indicating a recombination frequency of about

Physarum aZcA MUTANTS 43 TABLE 5

Progeny analysis of crosses between alc strains*

alc:

+ -

~~

Genotype Numb er+ fusA whi Cross 1s Cross 2

any any 1 0 1 + 19 22 1 1 13 13 2 + 34 23 2 1 44 11 Y2 + 39 4 Y2 1 14 1

Totals I@ 74

*All progeny clones, whether plasmodial or amoebal, were analyzed. The latter type of

Jr Cross 1 : CH987 matA3 matB3 fusAI whi+ a b 8 x CH967 matAI matBI fusA2 whi-I alc-IO. Cross 2: CH986 matA2 matB3 fusAl whi-I alc-8 x CH970 matA3 matBl fusA2 whi+ alc-11.

2 Progeny designated "-" were either plasmodia, or amoebae that differentiated to plasmodia at 30" within 10 days. 0 This and other crosses of CH967(fusAZ) with fusAl strains consistently gave an excess of

fusA.2 progeny and an unusually large number of fusAI/fusA2 progeny. The data are consistent with the assumption that strain CH967 is disomic for the fusA chromosome.

progeny were allowed to form plasmodia to score for whi and fusA.

2 X 3/1000 = 0.6% between aZcA8 and aZcAZO. We doubt that the three progeny are revertants, since aZcA8 reverts at a frequency of about and alcAlO at <5 X loe5 (see below). Recombination between aZcAlO and aZcAZi was also found to be quite low: aZcA+ progeny generally occurred at a frequency of 0.5% or less.

Effect of temperature on growth and differentiation of alcA amoebae: As has been noted, aZcA amoebae self much more frequently at high temperature (30") than at low (21 ") . We also observed that aZcA8 and alcAll amoebal colonies, unlike aZc+ amoebal colonies, enlarge more slowly at 30" than at 26". These effects of temperature on differentiation and colony growth were studied further with a standard "kinetics" procedure (YOUNGMAN et al. 1977). In this procedure, a set of replicate plates of amoebae is prepared at a particular time. At various subsequent times, cells are harvested from representative plates and assayed for total number of cells by microscopy and for number of plasmodia by replating under conditions (in this case 21 ") that inhibit further commitment to plasmodia but permit growth of small plasmodia to a visible size.

The effect of temperature on aZcA8 amoebae is shown in Figure 1A. At 21", commitment to plasmodium formation by the aZcA8 amoebae occurred at a frequency of about 10-5/amoebal cell. For comparison, wild-type (aZc+ gad+) amoebae self at a frequency of less than at all temperatures. At 26", the frequency for aZcA8 amoebae rose to about which shows the expected marked increase in plasmodium formation with increased temperature. The increased selfing seen at 30" was correlated with a decrease in amoebal growth rate at this temperature. The doubling time of aZcA8 amoebae at 26" was 9 hr; at 30", the doubling time gradually increased to about 34 hr. In con-

and at 30" to

44

I o7

I o6

I o5

F 2 lo4 3 U \ ln - - 8 lo3

I o2

IO'

C. L. TRUITT, C. S . HOFFMAN A N D C . E. HOLT

Time (days)

FIGURE 1.-Kinetics of growth and differentiation of &A amoebae. Spot cultures were set up with amoebae that had been pregrown at 21", In this figure and the remaining figures, open symbols represent total number of cells as determined in a hemacytometer, closed symbols represent numbers of assay plasmodia, and the horizontal dashed line represents the minimum detectable number of assay plasmodia. The symbols + and X in this figure represent numbers of viable amoebae as determined by the formation of amoeba1 plaques on assay plates. A) Effect of tcmperature of CH918 alcA8 amoebae; 21", (0, 0 ) ; 26", (U, m); 30", ( A , A ) . B) Com- parison of the growth and viability of LU911 a k A + (0, +) and CH918 aZcA8 (A, X) amoebae at 30". C) Comparison at 30" of CH918 aZcA8 (0, a), CH967 alcAlO (U,.), and CH970 alcAl1 (A, A).

trast, &A+ amoebae grow with a doubling time of about 8 hr at both temperatures (data not shown).

Besides having a lower growth rate at 30°, alcA8 cells at this temperature generally had an aberrant, enlarged appearance under the microscope. This raises the possibility that a large fraction of the alcA8 cells undergo the early stages of differentiation but fail to become committed to the plasmodial phase or to continue multiplying. Data consistent with this possibility were obtained in an experiment in which the number of cells determined by hemacytometer count was compared with the number of viable amoebae. The viable count of the &A8 amoebae at 30" was only about 20% of the hemacytometer count (Figure 1B). For comparison. the hemacytometer and viable counts of &A+ cells, which grew four-fold faster than akA8 cells at 30°, were about the same.

Physarum aZcA MUTANTS 45 The behavior of strains carrying aZcA8, aZcAlO and a lcA l l was compared

with the kinetics procedure. At 30", the three strains showed reduced growth compared to wild type (Figures 1B and IC), and their doubling times during the exponential phase did not differ significantly from one another (Figure IC) . Plasmodium formation, on the other hand, did differ significantly, with the aZcAll strain clearly producing plasmodia earlier than the other two strains.

Effect of temperclture on alcA8 plasmodial growth: It seemed possible that the aZcA8 mutation affected growth at 30" rather nonspecifically. This possibility was examined by comparing the growth of CH918(aZcA8) haploid microplasmodia and LU904 (gad-h aZcA+) haploid microplasmodia at 30". Both strains grew with a doubling time of about 24 hr and reached a maximum density of about 3 mg dry weight/ml. This indicates that the effect of aZcA8 on growth at elevated temperatures is limited to the amoebal phase.

Effect of temperature on alcA8 germination behauior: As the aZcA8 lesion results in thermosensitivity with regard to amoebal stability, we wished to determine if an aZcA8-mediated temperature effect was evident during the establishment of the amoebal state at either sporulation or germination. In one type of experiment. aZcA8 plasmodia were subjected to the usual conditions for inducing sporulation at 21", 26", and 30", and the resulting spores were germinated at 26". Spores formed at 21" and 26" gave rise to essentially identical (13% us. 20%) percentages of plasmodial progeny. Neither aZcA8 nor aZcA + plasmodia sporulated at 30". In another type of experiment, spores formed at room temperature (about 24") were germinated at 21", 26" and 30". Both amoebal and plasmodial progeny were found at 21 " and 26", and the ratio of the two progeny types was identical. At 30", a small number of plasmodial progeny appeared on spore germination plates. No amoebal plaques appeared; none were expected, because control experiments showed that &A8 amoebae do not form visible clones at 30", apparently due to their slow growth and viability at this temperature (Figures 1B and IC). Thus, we were unable to find evidence that the tendency of aZc spores to release plasmodia is temperature sensitive.

alcA reuertants: Four phenotypic manifestations of the aZcA mutations are germination as plasmodia, enhanced amoeba1 selfing, reduced amoebal growth and reduced mating ability. As discussed above, the first two phenotypic properties cosegregated. Qualitative observations on the growth of amoebae in differentiation tests of the aZcA x &A+ progeny showed that selfing and reduced amoebal growth also cosegregated. Thus, these results suggested that at least the first three of the abnormalities of an aZcA mutant are the result of a lesion in a single gene. Nevertheless, the possibilities of multiple tightly-linked mutations or of a deletion of several genes existed. For this reason, we set out to isolate revertants of aZcA amoebae which were wild type with respect to amoebal growth at 30". Suspensions were made from six isolated colonies of CH918 aZcA8 amoebae which had been grown at 21". Spot cultures of these suspensions were made with about 5 x lo3 cells/culture. The cultures were allowed some further growth to about lo4 cells each and then transferred to 30", at which temperature only a 200-fold increase in the number of &A8 amoebae occurs

46 C . L. TKUITT, C. S. HOFFMAN A N D C . E. HOLT

before growth stops. The cessation of growth occurs after six to seven days of incubation when there are about 2 x I O G amoebae per plate. At this time there were no visible amoebal plaques. (In contrast, &A+ amoebae form small, clearly visible plaques after four days at 30"). Occasional amoebal plaques (averaging two per plate) did appear on the bacterial lawns by 11 days. This number of plaques represents a frequency of revertants of about 1 0-6 (ttvo/plate divided by 2 x loF amoebae/plate) . Six independently isolated revertants were purified and used in subsequent analyses.

The six revertants were subjected to the standard kinetics procedure to determine their growth and differentiation at 30". Typical results are shown in Figure 2. In all cases, the revertant amoebae grew at a rate indistinguishable from &A+ amoebae and exhibited no differentiation to plasmodia in six days. In addition, four of the revertants were tested to see whether normal mating ability and spore germination had been restored. The tests involved mixing each of the revertants with &A8 amoebae carrying different mrrtA and mutB alleles,

I I I I

A lo7 -

0 2 4 6 Time (days)

FIGURE 2.-Growth of revertant amoebae. Spot cultures of two independent revertant amoeba1 strains and of &A+ and &A8 amoebae as controls were incubated at 30" and assayed as in the experiments of Figure 1. LU911 a1cA-f- (0, 0 ) ; CH918 &A8 (U, a); two &A+ revertants (A, A), (V, W.


sporulating the resulting plasmodia, and examining the germination of the spores and the properties of the progeny amoebal clones. The plasmodia were in each case crossed diploids, which shows that the four revertants had regained mating ability. (In control experiments, comparable alcA8 x alcA8 mating mixtures produced only parental haploid plasmodia or haploid heterokaryon plasmodia, never diploid plasmodia.) Moreover, the spores from each of the revertant X alcA8 plasmodia germinated as amoebae only, which shows that the revertants had regained a dominant allele conferring normal germination behavior. As expected, half the progeny of each of the plasmodia differentiated to plasmodia at 30" and half did not. In summary, the growth temperature sensitivity of aZcA8 amoebae was lost at a frequency of about and this loss was always accompanied by a loss of the other three &A8 characteristics. Similar results were obtained with aZcAlO. The results indicate that the alcA phenotype can result from a lesion in a single gene.

Effect of extracellular inducer on alcA amoebul differentiatidn: Cultures of amoebae carrying gad mutations normally begin differentiation at a critical cell density characteristic of the strain (ADLER and HOLT 1977) and can be induced to differentiate at much lower densities by an extracellular inducer (YOUNGMAN et al. 1977). It has been proposed that the critical density is the one at which there has been a sufficient accumulation of inducer to trigger the differentiation (YOUNGMAN 1979).

The time course of plasmodium formation in an akA8 amoebal culture differs markedly from that in a gad amoebal culture. In an alcA8 culture at 30", the number of plasmodia was a constant proportion of the number of amoebae after a lag (Figure 1A) . This result was obtained whether cultures were started at IO" amoebae/culture (Figure IA) or at IO3 or 5 x I O 4 amoebae/culture (data not shown). The length of the lag before the plasmodial curve paralleled the amoebal curve was reduced by pregrowth of the amoebae at 26" rather than at 21" as in the experiment of Figure IA. These results are in sharp contrast to the pattern of differentiation in gad amoebae, which display a dramatic, rapid rise in the number of committed cells, with a doubling time that is much shorter than that of the amoebal growth curve.

To determine whether alcA8 differentiation is inducible, we mixed akA8 amoebae with a four-fold excess of CH814, an &A+ strain carrying the same matA and matB alleles and known to be an efficient inducing strain. The formation of plasmodia in the mixture was assayed. The number of plasmodia, all of which were derived from the alcA8 strain, was no higher in the mixture than in the control culture of &A8 amoebae alone (Figure 3). The induction experiment was conducted with the inducing strain CH814 and the responding strain mixed with one another because induction of differentiation is stronger with this arrangement than with the strains separated by filters (YOUNGMAN, SMITH and HOLT, unpublished results). In a control to check the inducing ability of CH814, the CH814( matAS) amoebae and the responding LU904( matAh) amoebae were separated by filters, since matA3 and gad-h amoebae can mate. The control data show that CH814 amoebae are indeed capable of inducing

48 C. L. TRUITT, C. S. HOFFMAN AND C. E. HOLT

I o7

I o6

I o5

S lo4

0" lo3

I o2

I 0'

??

V \ In - -

I 1 I I

*&-

FIGURE 3.--Inability of &A+ amoebae to induce selfing of aZcA amoebae. The foIlowing cultures were prepared, incubated at 308", and assayed as in Figure 1: CH814 &A+ amoebae at an initial density of IO4 cells/culture ('0, B); CH814 and CH918 &A8 amoebae at a ratio of 4:l and an initial total density of I@ cells/culture (A, A); and CH918 amoebae at the same initial density, that is 2 x IOs/culture (0, 0 ) . As a control some of the cultures of CH814 &A+ amoebae were used as inducing cultures for sparse cultures of LU904 gad-h amoebae. The gad-h amoebae were inoculated onto a pair of sterile (0.2 fim pore diameter) NucIepore filters at an initial density of 100 cells/culture and were incubated at 30". At 24 hr (arrow), half these gad-h cultures were transferred onto the C,H814 spot cultures and the other half, onto spot cultures containing bacteria alone. Incubation was continued at 30", and the number of gad-h assay plasmodia was determined in the induced (X) and uninduced (+) cultures at intervals (YOUNGMAN et al. 1977).

differentiation in gad-h amoebae. Thus, differentiation of aZcA8 could not be induced.

Possibly alcA8 amoebae are actually sensitive to inducer, but overproduce it to such an extent that they are saturated with it even at very low cell densities. If this is the case, aZcA8 amoebae should induce differentiation in sparse cultures of gad-h amoebae better than the parental alcA+ amoebae. We found. however, that under conditions in which the inducing alcA8 culture was itself differ- entiating, its stimulation of gad-h amoeba1 differentiation was comparable to


the stimulation achieved by the aZcAf parent (Figure 4). Thus, inducing ability in &A8 amoebae appears to be normal.

These results, taken together, indicate that selfing in aZcA8 cells is independent of the effects of an inducer.

Interaction between the alcA8 mutation and mutations that block diflerentia- tion: Mutations (“apt” or ‘‘npf”) that block asexual (gad-mediated) and sexual plasmodium formation are known. To determine whether differentiation of aZcA amoebae involves the same pathways of plasmodium formation, nZcA strains carrying apt or npf mutations were constructed and their differentiation properties determined.

The recessive aptAl lesion (WHEALS 1973) prevents mating, as well as selfing by all gad mutants tested (SHINNICK 1978; SHINNICK, ANDERSON and

FIGURE 4.-Comparison of alcA and a k A + amoebae as inducers of gad-h selfing. Spot cultures of CH918 elc8 amoebae (0) or LU911 &A+ amoebae (O), a t initial densities of 104 amoebae/ culture, were incubated at 26”. At the same time, LU904 gad-h amoebae were inoculated at 100 cells/culture onto pairs of Nuclepore filters and incubated at 26”. At 36 hr (arrow), the gad-h cultures were transferred onto either alcA + cultures, &A8 cultures, or control plates containing only bacteria. The numbers of assay plasmodia in the gad-h cultures a t various times after transfer are represented by closed symbols corresponding to the bottom, inducing cultures: alcA f ( ) ; alcA8 (m) ; uninduced control (A). (Measurement of the numbers of aZcA8 assay plasmodia in the &AI inducing cultures demonstrated that aZcA8 amoebae differentiated normally under these conditions [data not shown] .)

50 C. L. TRUITT, C. S. HOFFMAN AND C. E. HOLT

HOLT, in preparation). A strain possessing the apt.41 lesion without any accom- panying gad mutation was crossed with an alcA8 amoebal strain (Table 6). AS expected of an aZcA+ X aZcA8 cross, all spores germinated as amoebae. Half the aptA+ progeny selfed at 30" and half did not, presumably reflecting the segregation of alcA8 and aZcAf alleles (Table 6 ) . None of the aptA1 progeny selfed. Five of the aptA1 progeny were crossed with an aZcA+ ap tA f amoebal strain. Three of the five gave selfing amoebal clones among their progeny, demon- strating the presence of the aZcA8 mutation in these nonselfing, aptAl progeny. Thus, aptA1 blocks alcA8 amoebal differentiation.

The growth of alcA8 amoebae is inhibited at 30" (Figure IB). To determine whether an aptA1 strain possessing the alcA8 mutation would still exhibit this particular alcA8 effect, the growth of the five aptA1 progeny analyzed above was measured at 30". As expected, these progeny failed to differentiate into plasmodia. None of the progeny grew at the greatly reduced rate characteristic of aZcA8 aptA+ amoebae. Instead, the two alcA+ aptA1 strains grew identically to the aZcA+ aptA1 parent, and the three &A8 aptA1 progeny grew at a rate intermediate between the alcA+ aptA1 and the aZcA8 aptA+ parental strain rates (Figure 5 ) . These results show that the aptA1 lesion not only prevents differentiation by alcA8 amoebae possessing it, but also largely reverses the aZcA8-mediated inhibition of cell growth at 30". In addition, there was a three- fold enhancement of cell viability in aZcA8 aptA1 amoebae grown at 30" when compared with the viability of &A8 aptA+ amoebae grown under the same conditions (for example, 53% us. 18% viability after 24 hr at 30" ) .

The mutation npfA1 is the only other npf or upt mutation available that has been separable from n a t A by recombination (WHEALS 1973; ANDERSON and DEE 1977). The np fA l lesion abolishes o r greatly reduces selfing at 26" and 30" in gad-h strains (ANDERSON and DEE 1977) and in strains carrying most other gad mutations (gad-1,2,3, 4,5,~~,~4,~5,l6), but not in strains carrying the mutations gsd-6 and gad-13 (SHINNICK 1978). The np fA l mutation has little or no

TABLE 6

Progeny analysis of an alcA+ aptAI x alcA8 aptA+ cross'

Number of progeny Genotype Koonselfinw Selfing

Experiment I mntAl aptA+ m a t A l aptAI matA3 aptA+ matA3 aptAI

t aptA+ 1- aptAI

Experiment I1

5 13 4 5

14 37

21 0

* Parental strains: LU865 matA1 matB1 fusA l whi+ aptAl aZcA+ x CH918 matA3 matB3

t matA type not determined. f u sA2 whi-l aptA+ alcA8.

c 7 -

a W P Q) 0 E

I o7

I o6

I o5

I o4

Physarum aZcA MUTANTS 51

1 I I I I I I J 0 2 4 6

Time (days) FIGURE 5.-Growth of alcA8 aptAI amoebae. Growth at 30" of CH918 akA8 aptdf (U),

LU865 alcA+ aptAI (0) and an alcA aptA2 recombinant of these strains ( A ) was measured by hemacytometer counts. Two other alcA8 aptdl progeny were also tested and gave comparable results.

effect on sexual differentiation by ordinary heterothallic strains (matAl,2,3,4) (ANDERSON and DEE 1977).

The effect of the npfAl mutation upon differentiation by aZcA8 amoebae was analyzed by crossing CH918 matA3 alcA8 npfA+ by LU869 matAl alcA+ npfAl (Table 7). Half the progeny were aZcA8 and half were &A+, based upon the ability to differentiate to plasmodia at 30". The npfA allele present in each of 50 matA3 progeny was determined by a complementation test (see

TABLE 7

Progeny analysis of an npfAl alcA+ x npfA+ alcA8 cross*

Number of progeny Genotype Nonselfing Selfing

matAI matBl matAI matB3 matA3 matB1 matA3 matB3

matA3 npfA+ matA3 npfAI

10 9 8

18 Total 45

12 13

Total 25

15 8

11 15 49

11 14 25

* Parental strains: LU869 matAI matBI whi+ fusA2 npfA1 alcA+ X CH918 matA3 matB3 whi-1 fusA2 npfA+ alcA8.

52 C. L. TRUITT, C. S. HOFFMAN A N D C . E. HOLT

MATERIALS AND METHODS). The npfAl mutation was present in 13/25 aZcA+ progeny and in 14/25 &A8 progeny. Thus, EpfA recombines freely with aicA. As the alcA8 npfAl progeny differentiated as expected of alcA8 amoebal strains, the npfAI mutation does not prevent differentiation by alcA8 amoebae. This result in contrast with that obtained with aptAl , suggests that selfing by alcA8 amoebae begins at a later step in a pathway, compared to the point at which np fA acts, or takes part in a different pathway.

DISCUSSION

The three aZc mutations that we completely analyzed fall into one complementation group (a lcA) , and a fourth, alc-9, also appears to fall into the same group. These results suggest that there are few genes, perhaps only one, that regulate both the re-establishment of the amoebal state at sporulation/germination and the stability of the amoebal phase of growth, once it is established. It is, of course, possible that other alc genes exist that mutate more rarely or that cannot produce conditional o r “leaky” mutations that allow the isolation of occasional alc amoebal strains. The alc mutations that could not be analyzed genetically because they never produced alc amoebae at spore germination could represent mutations in other aic genes, the wild-type products of which are absolutely required for normal spore germination. Given the wide range of frequencies (0.001 to 0.5) of amoebal progeny arising from spores of the various alcA mutants, however, it seems possible that the uncharacterized alc mutations are simply particularly severe mutations in the aicA gene.

The 31 alc plasmodia that we isolated in the mutant hunt came from a total of 2688 spot cultures of unmutagenized amoebae (Table 2). These cultures grew to a maximum density of about 2 x I O 7 amoebae per plate after five to six days of incubation, and most of the alc plasmodia became visible on the plates after two to three weeks of incubation. Thus, the ratio of plasmodia isolated to the number of amoebae was about 5 x The frequency of alc amoebae in the population is likely to be several orders of magnitude higher than this, as alc amoebae become committed to the plasmodial form at a low efficiency (Figure 1 ) . An accurate measurement of the mutant frequency would require a number of additional pieces of information including knowledge of the differentiation efficiencies of aic mutant amoebae that arise at different times during the incubation of spot cultures.

The recessive aptAI mutation prevents all types of differentiation from amoebae to plasmodia that have been looked at: gad- or &A-mediated selfing or matA-controlled mating (WHEALS 1973; SHINNICK 1978; and present work). The effect of the aptAI lesion an aicA8 amoebae appears similar to its effect upon gad-h amoebae. The aptAI mutation was initially isolated by screening for nondifferentiating gad-h plaques (WHEALS 1973). DAVIDOW and HOLT (1977) found that sequential enrichment fo r amoebae in a gad-h culture initially containing a minority of gid-h aptAI amoebae indeed selected for the aptAI-containing gad-h amoebae. Thus, the aptAI mutation does not act simply


by preventing some terminal step in gad-h differentiation to plasmodia, with resulting cell death, but must actually stabilize gad-h cells in the amoebal phase of growth, resulting in an enhancement of amoebal growth or survival like that seen in &A8 aptAI amoebal cultures.

The lowered growth rate of aZcA amoebae at 30" may be due solely to a high rate of amoebal cell death, with a normal division time for the remaining viable amoebae. The difference between the total number of aZcA amoebae during growth at 30" and the number of viable amoebae is not inconsistent with a relatively high rate of cell death (but less than 50%), coupled with a normal division time for the viable amoebae. One possibility is that a rather high percentage of aZcA amoebae initiate some step in differentiation to plasmodia at 30" but, due to unsuitable intracellular or environmental conditions, only a small percentage successfully make the transition, and the other cells die in the attempt. In this scheme, the aptAl lesion could partially reverse the aZcA8- mediated inhibition of amoebal growth at 30" by preventing a relatively early step in aZcA8 amoebal differentiation, thus stabilizing aZcA8 cells as viable amoebae. Indeed, the percentage of viable aZcA8 ap tAI amoebal cells as a function of total cells at 30" was found to be significantly (three-fold) higher than the percentage of viable aZcA8 aptA+ amoebal cells grown under the same conditions.

The reduction in mating efficiency displayed by alc amoebae could arise from interference with any of the several steps of mating. One of the steps, nuclear fusion, is a process that occurs only in amoebae; nuclei in a multinucleate plasmodium do not fuse with one another. Possibly then, the poor mating ability of alc amoebae results from their nuclei having acquired prematurely the poor fusion ability of plasmodial nuclei.

The question of which step(s) during sporulation and germination is in- fluenced by the alcA+ gene product is an interesting one which has yet to be answered. One line of evidence suggests that the ratio of amoebal to plasmodial progeny emerging from aZcA spores is determined before or during sporulation, rather than at spore germination: there is up to a 10-fold variation in the amoebae to plasmodia ratio in the spores from one sporulation plate as compared with those from another, but reproducible results are obtained at germination with the spores from a single sporulation plate. The aZcA product is presumably synthesized before the meiotic division, since all the spores from an a2cA +,/&A heterozygote germinate as amoebae, Function of the product could, however, be delayed until germination. Further genetic and physiological analyses aimed at determining the timing of action of the &A+ gene product in the re-establishment of the amoebal state are underway.

The effect of an alcA mutation upon maintenance of the amoebal state, once established, is somewhat better characterized, although the fundamental question of its mode of action remains. Several differences are evident in selfing mediated by gad us. aZcA mutations: (1) The npfAl mutation, which prevents differentiation from amoebae to plasmodia in most gad mutants (WHEALS 1973; SHINNICK 19781, does not prevent the differentiation of aZcA8 amoebae. (2) All

54 C . L. TRUITT, C . S. HOFFMAN AND C. E. HOLT

aZcA amoebal strains tested exhibit a low frequency of differentiation to plasmodia (<I %) , whereas gsd-h amoebae can differentiate very efficiently ( > l o % ) (YOUNGMAN et aI. 1977). (3) Differentiation by aZcA8 amoebae is not affected by the presence of inducer, whereas gad-h amoebae are very sensitive to its presence (YOUNGMAN et al. 1977). Possibly, the aIcA f gene product acts along a different pathway of differentiation control in amoebae than does the gad-h+ gene product. Alternatively, the alcA+ product might act along the same pathway, beyond the steps at which the gad-h+ product, the inducer, and the npfA+ product act, but before the step at which the aptA+ product acts. The analysis of patterns of differentiation from amoebae to plasmodia in aZcA gad-h double mutants may help to distinguish between these possibilities.

We thank ROGER ANDERSON for helpful suggestions concerning the npfA and aptA analyses and for providing us with the necessary strains. This work was supported by National Science Foundation grant #7923507-PCM to C. E. H. C. L. T. was supported by American Cancer Society and Chaim Weizmann Postdoctoral Fehwships. C. S. H. was supported by the M.I.T. Undergraduate Research Opportunities Program.

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ADLER, P. N. and C. E. HOLT, 1974 Genetic analysis in the Colonia strain of Physarum polycephalum: heterothallic strains that mate with and are partially isogenic to the Colonia strain. Genetics 78: 1051-1062. ~ , 1975 Mating type and the differentiated state in Physarum polycephalum. Develop. Biol. 43: 240-253. - , 1977 Mutations affecting the differentiated state in Physarum polycephalum. Genetics 87: 401420.

Analysis of the amoebal-plasmodial transition in Physarum polycephalum. Ph.D. Thesis, Univ. of Leicester. --- , 1979 Complementation of amoebal- plasmodial transition mutants in Physarum polycephalum. Genetics 91 : 409419.

ANDERSON, R. W., D. J. COOKE and J. DEF, 1976 Apogamic development of plasmodia in the Myxomycete Physarum polycephalum: A cinematographic analysis. Protoplasma 89 : 29-40.

ANDERSON, R. W. and J. DEE, 1977 Isolation and analysis of amoebal-plasmodial transition mutants in the Myxomycete Physarum polycephalum. Genet. Res. Camb. 29: 21-34.

ANDERSON, R. W. and C. E. HOLT, 1981 Revertants of selfing (gad) mutants in Physarum Dolrcephalum. Developmental Genetics 2 : 253-267.

COOKI:, D. J. and J. DEE, 1975 Methods for the isolation and analysis of plasmodial mutants in Physarum polycephalum. Genet. Res. Camb. 24: 175-187.

DAVIDOW, L. S. and C. E. HOLT, 1977 Mutants with decreased differentiation to plasmodia in Physarum polycephalum. Molec. Gen. Genet. 155: 291-300.

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ANDERSON, R. W., 1976

Physarum alcA MUTANTS 55 GORMAN, J. A. and A. S. WILKINS, 1980 Developmental phases in the life cycle of Physarum

and related myxomyces. pp. 157-202. In: Growth and Differentiation in Physarum polycephalum. Edited by W. F. DOVE and H. P. RUSCH. Princeton University Press, Princeton, N. J.

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SHINNICK, T. M. and C. E. HOLT, 1977 Analysis of an mt-linked mutation affecting asexual plasmodium formation in Physarum. J. Bacteriol. 131: 247-250.

SHINNICK, T. M., 1978 SHINNICK, T. M., D. J. PALLOTTA, Y. R. JONES-BROWN, P. J. YOUNGMAN and C. E. HOLT, 1978

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Corresponding editor: S. L. ALLEN

HOLT, C. E., A. HUTTERMAN, H. HEUNERT and H.-K. GALLE, 1979

Ph.D. Thesis, Massachusetts Institute of Technology.

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YOUNGMAN, P. J., R. W. ANDERSCIN and C. E. HOLT, 1981

Documents

A ALCA, THE FORM EXPRESSED IN PHYSARUM POLYCEPHALUM · A GENE, ALCA, AFFECTING THE LIFE CYCLE FORM EXPRESSED IN PHYSARUM POLYCEPHALUM CHRISTINE L. TRUITT1, CHARLES S. HOFFMAN2 AND