Copyright 8 1988 by the Genetics Soaety for America

motA1552, a Mutation of Dictyostelium discoideum Having Pleiotropic Effects on Motility and Discoidin I Regulation

Samuel C. Kayman, Richard Birchman and Margaret Clarke Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

Manuscript received July 15, 1987 Revised copy accepted November 9, 1987

ABSTRACT The Dictyostelium dkcoideum mutant MC2 exhibits temperature-sensitive growth, temperature-

sensitive motility, and temperature induction of discoidin I synthesis. These three phenotypes of MC2 were not separated in the genetic experiments reported here. They were therefore assigned to the mutation motA1552, which was mapped to linkage group I1 by segregation analysis and by analysis of mitotic recombinant diploids. In one motA1552 strain, loss of motility preceded accu- mulation of discoidin I by 3 hr, indicating that discoidin I is not involved in generation of the motility defect. Expression of motA1552 phenotypes varied both among strains carrying the mutation, and among different clones of a particular strain. MC2 and its derivatives displayed elevated levels of recombination between whiA and acrA on linkage group 11, and yielded highly unstable mutations at the acrA locus. Accumulation of large amounts of discoidin I during axenic growth of strain AX3 was found to depend on the presence of a second linkage group I1 mutation, daxAI551. This mutation was already present in the strain mutagenized to isolate motA1552, complicating explication

I .

of motA1552 action. u

D URING vegetative growth, the cellular slime mold Dzctyostelzum discoideum lives as unicellular

amoebae. This organism also manifests a develop- mental phase leading to spore formation, in which amoebae form aggregates that function as multicel- lular organisms. A variety of signals and genetic components have been implicated in the control of development and the intricate regulation of gene expression that is involved. [See GODFREY and Suss- MAN (1982), LOOMIS (1982, 1987), and SCHAAP (1986) for reviews.] Among the proteins synthesized early in development are the discoidins. Discoidin I pro- teins are encoded by three coordinately regulated structural genes and normally reach their highest level in aggregating cells (reviewed by KIMMEL and FIRTEL 1982). Discoidin I has some homology to fibronectin; it appears to function in cell-substratum adhesion and in the formation of cell streams during aggregation (SPRINGER, COOPER and BARONDES 1984; CROWLEY et al. 1985).

The complex regulation of the discoidin genes is under investigation. Induction of discoidin I (and at least two other early developmental proteins) actually precedes development, occurring during exponential growth. This induction is under the control of a system that monitors both cell and bacterial (food) densities (CLARKE, KAYMAN and RILEY 1987). Ter- mination of discoidin I transcription may be mediated by formation of cell-cell contacts, and elevation of intracellular CAMP levels (BERGER et al. 1985; WIL- LIAMS, TSANG and MAHBUBANI 1980). Two loci iden-

Genetics 118: 425-436 (March, 1988)

tified by mutations that block the accumulation of discoidins I and I1 under developmental conditions have been mapped to linkage groups I1 and 111; a third regulatory locus, identified by a mutation that suppresses the linkage group I11 mutation and causes high levels of discoidin during growth on bacteria, was tentatively mapped to linkage group IV (ALEX- ANDER, CIBULSKY and CUNEO 1986). Based on the behavior of double mutants, these loci were ordered along a regulatory pathway for discoidin synthesis during development.

In this paper, two other mutations that affect discoidin I regulation are characterized. One is motA1552, initially characterized in the strain MC2 as responsible for temperature sensitive growth (Tsg- ) and temperature-sensitive motility (Mot‘”) (CLARKE 1978; KAYMAN, REICHEL and CLARKE 1982). MC2 also initiates synthesis of discoidin I and accu- mulates this protein to extremely high levels when shifted to restrictive temperature in the presence of a food source (BISWAS, KAYMAN and CLARKE 1984); this phenotype is designated Dis’”. Mapping data that support assignment of the Disfs phenotype to mot- A1552 are presented here, and the interdependence of the Mot” and Dis‘” phenotypes is addressed. MC2 was also found to display a number of genetic inst- abilities; possible explanations for these instabilities are considered.

The second mutation is daxA1551, identified here as responsible for the high levels of discoidin I seen during growth of AX3 on axenic (soluble nutrient)

426 S. C. Kayman, R. Birchman and M. Clarke

TABLE 1

Genotypes of haploid strains used in this work

Strain Parent acr ax& bsg bwn cob q c d m man mot nys oaa spr trg whi Originb

AX3 HK12 HU25 HU49 HU1136 MC2 MC3 MC204 MC279 MC288 MC293 MC329 MC335 MC338 MC345 MC347 MC349 MC445 MC487 MC493 MC5 13 MC5 14 MC5 17 NP2 TS12 XP55

NC4 TSl2M X22" DP4c DU1461 AX3 AX3 DMC 1 DMC68 NP2 MC288 DMC 105 DMC105 DMC105 DMC105 DMC105 DMC95 MC349 DMC 149 MC487 MC514 MC2 DMC 150 AX3 NC4 DP746'

+ A201 AI

A3 71 + + +

A1 +

A1551 A1551

+ +

A1551 +

A1551 +

A I553 + + +

A1552 + + + +

BI,CI + + + + + + +

C l d 8500 BI,CI + BI,CI +

+ A5 BI,CI + B1,CI + BI,CI + B1 + BI + BI,CI + BI + BI,CI + BI,CI + BI,CI + BI ,CI? + BI,CI? + BI,CI + BI,CI + BI,CI B500 BI ,CI +

+ + + A5

-t + +

AI AI + -t +

AI -t +

AI + + +

AI AI AI + + + + -t + + +

+ +

A352 A358

+ + + +

A358 + +

A358 + + +

A358 A358 A358

+ + + + + + + +

+ A1 + +

AI + +

A5 + + + + + + + +

AI AI A5 A5 + +

AI +

AI A5

A1551 + + + + + + +

A1551 A2 A1551 + A1551 +

+ AI A1551 + A1551 + A1551 +

+ + + +

A1551 + + +

A1551 + A1551 + A1551 + A1551? + A1551? + A1551 + A1551 + A1551 A2 A1551 +

+ + + +

~~

+ + A I + + C210 + + + + + A I + + + A I + + A I +

A1552 + AI + -1553 + AI +

+ + + + A1552 + A1 +

+ + A I + + 81551 AI + + + + + + B1551 + + + B1551 AI + + B1551 + + + + AI AI

A1552 + AI + A1552 + AI +

+ + A I ? + + + A I ? +

A1552 + A1 + A1552 + AI + A1552 + AI +

+ + A I + + + + + + + + +

+ + 2 0 1 2 AI 3

DI2,E13 A I 4 E13 + 4 + B513 5 + + 6 + + 6

D l 2 AI 1 + + 1

AI + 1 AI + 1 AI + 1 AI + 1 AI + 1 AI + 1

AI,E13' + 1 + + 1 + + 1

D l 2 AI 1 D l 2 AI 1 + + 1 + + 1 + + I

AI + 7 D l 2 AI 8 + + 9

Phenotypes of all mutations are summarized in NEWELL (1982), except for mot and dm mutations, which are described here and in KAYMAN, REICHEL and CLARKE (1982). A " + " denotes the wild-type allele.

Parents of diploids not previously described are: DMCl (MC201 X XP55), MC2Ql has a complex geneology, including NC4, AX3, TS12, and M28 and their derivatives; DMC68 (HU49 X MC2); DMC95 (HK12 X MC279); DMC105 (HU49 X MC293); DMC144 (MC445 X TS12); DMC149 (MC2 X MC204); DMC150 (HU1136 X MC2).

Nomenclature for the axenic mutations is according to CLARKE and KAYMAN (1987). 1) This work; 2) LOOMIS 1971; 3) KASBEKAR, MADICAN and KATZ 1983; 4) WELKER and WILLIAMS 1980; 5) WELKER, METZ and WILLIAMS

1982; 6) KAYMAN, REICHEL and CLARKE 1982; 7) KESSIN, WILLIAMS and NEWELL 1974; 8) KATZ and SUSSMAN 1972; 9) RATNER and NEWELL 1978.

The complex genetic background of this strain does not include AX3 or another axenic strain. Established by axenic growth characteristics of segregants from DMC150 that carried acrA371 and consistent with its geneology. Established by linked markers axeBl for tsgAl and g r A l for tsgEl3.

medium (ROSEN et al. 1973); this phenotype is des- ignated Dis"". The ability to grow axenically is itself a mutant phenotype; wild-type D. discoideum cells require a bacterial food source. Mutations involved in axenic growth have been mapped to linkage groups I1 and I11 (GINGOLD and ASHWORTH 1974; WILLIAMS, KESSIN and NEWELL, 1974). The existence of dux- A1551 complicates the study of motA1552, because motA1552 was isolated in the presence of d 4 1 5 5 1 .

MATERIALS AND METHODS

Growth of cells. Strains of D . discoideum were maintained on silica gel, and working cultures were passaged on Klebsiella aerogms or HL5 axenic medium as described (KAYMAn~and CLARKE 1983). Growth in bacterial suspension culture has been described (CLARKE, KAYMAN and RILEY 1987). Genotypes and origins of strains used in this work are presented in Table 1; assignment of newly isolated acr and nys mutations to specific loci was by complementation

test. Development of bacterially grown cells in suspension was performed as described (CLARKE, KAYMAN and RILEY 1987).

Genetic manipulation and scoring of phenotypes. Ge- netic analysis in D . discodeurn makes use of parasexual fusion of haploid amoebae. Methodologies for production and segregation of diploids, and for scoring of standard genetic markers have been described (NEWELL 1982; WELKER and WILLIAMS 1982a). The restrictive temperature used was 27 f 0.2". Axenic strains were more sensitive to nystatin than wild type, requiring use of nystatin at 80 pgl ml, the lowest level reported by SCANDELLA, ROONEY and KATZ (1980).

The Tsg- phenotype was scored by stab test on bacterial lawns (CLARKE 1978) or by determination of efficiency of plating (EOP) at restrictive temperature. For recombinant haploids, the absence of parental tsg mutations was deter- mined in complementation tests by isolation of Tsg+ dip- loids with motA1552 or tsgD12 strains.

The MotU phenotype, severe loss of cell motility at restrictive temperature, was scored as previously described (KAYMAN, REICHEL and CLARKE 1982). Briefly, small clumps

Analysis of motA1552 427

FIGURE 1.-Complementation of Mot" phenotype of mot41552. Bacterial suspension cultures were passaged and incubated at permissive and restrictive temperatures for 12 hr. Cultures were diluted to 1 X lo3 cells per ml and 4 ml were incubated on gold-coated cover slips for 2 hr at the same temperature. Darkfield illumination was used to visualize the samples. Short tracks at 27" compared to those at 21" is indicative of the MotU phenotype. The sharply defined tracks of MC2, compared to the fuzzy tracks o f Hul l36 and DMCISO, is a recessive phenotype of axenic strains (KAYMAN and CLARKE 1983). Tracks of most strains were sharper at 27" than at 21". a, MC2, 21"; b, HUI 136, 21"; c, DMC150, 21'; d, MC2, 27"; e, HU1136, 27"; f, DMC150, 27" (magnification, X 26, bar = 0.5 mm).

of colloidal gold particles are deposited on a glass cover slip, and cells are then allowed to migrate across the lawn of particles. Cells collect the gold as they move, recording their paths as cleared tracks. Suspension cultures were inoculated and grown for at least 24 hr at 21" and then shifted to 27" for 12-18 hr before cells were taken for assay.

The Dis" phenotype, accumulation of discoidin I to high levels at 27" by cells in the presence of bacteria or axenic medium, was scored by SDS polyacrylamide gel electro- phoresis on samples prepared as previously described (CLARKE, KAYMAN and RILEY 1987). The intensity of the Coomassie-stained discoidin I band was monitored visually. Bacterial suspension cultures were used to ensure that the growth state was uniform throughout the population. Cul- tures were maintained at densities below those necessary for density-dependent induction of discoidin I (CLARKE, KAYMAN and RILEY 1987).

The Disa phenotype, discoidin I accumulation to devel- opmental levels during axenic growth, was similarly scored by SDS polyacrylamide gel electrophoresis. Cells growing in HL5 from newly started (grown for three generations) or established (passaged once or many times) cultures at densities between 5 x lo5 and 1 x lo7 gave qualitatively similar results.

Quantitation of translocation by analysis of videotape or photographic records has been described (CLARKE and KAYMAN 1987), as has detection of discoidin I by immu- nofluorescence and immunoblot (CLARKE, KAYMAN and RILEY 1987).

When diploids were scored for Mot or Dis phenotypes, the cell population was plated clonally under permissive conditions, and ploidy was confirmed by examination of spore size (diploid spores are larger than haploid spores).

Electrophoresis: Polyacrylamide gel electrophoresis in SDS was carried out using gels containing 12% acrylamide and the discontinuous buffer system described by LAEMMLI (1970).

RESULTS

Mapping of motA1552: The motility mutant MC2 manifests three phenotypes that had been tentatively assigned to a single mutation, motA1552: temperature sensitive growth (Tsg- ), temperature sensitive cell motility (MottS), and temperature induction of dis- coidin I (Dis"). This assignment was based on analysis of partial Tsg+ revertants of MC2, which also par- tially recovered normal motility and discoidin I reg- ulation (KAYMAN, REICHEL and CLARKE 1982; BISWAS, KAYMAN and CLARKE 1984). The data reported here support the conclusion that a single mutation is responsible for all three phenotypes.

Complementation analysis was carried out in sev- eral motA1552lmotA + heterozygotes. These diploids were scored for all three mutant phenotypes. Typical data from one diploid, DMC150 (MC2 X HU1136), are presented for the Mot and Dis phenotypes in Figures 1 and 2, respectively. Because motA1552 was isolated in the axenic genetic background, we tested diploids that were homozygous, as well as diploids heterozygous, for this background. The mutant phenotypes were not expressed in any diploid ex- amined. Thus, the Tsg-, Motts, and Dids phenotypes were all recessive.

motAI552 was mapped to linkage group I1 by examination of haploid segregants from several dip- loids heterozygous for motAI552. Analysis of haploid segregants of DMC150 (Table 2) demonstrated that the MottS and Dists phenotypes cosegregated with the Tsg- phenotype and that motAI552 segregated in-

428 S. C. Kayman, R. Birchman and M. Clarke

TABLE 2

Mapping of motAI552 in DMC150

Linkage group' Strains or

phenotypes Mot" Disb I I1 111 1v VI VI1 .. MC2 HU1136 + + cycA 1 ~ A 3 7 1 whiB513 hnAl m a d 2 bsgB500

tS + tS + + - + - + - + - + - + - TSG- 29 0 29 0 0 29 29 0 29 0 21 8 14 15 13 16 TSG+ 0 67 0 67 0 67 0 67 30 37 43 24 47 20 27 40

Only markers scored in this cross are indicated. See Table 1 for complete genotypes. Segregants were selected on cycloheximide-

D The Mot- phenotype is loss of motility at 27", scored by the gold-particle track assay. b The Dis" phenotype is production of large amounts of discoidin I at 27", scored by SDS polyacrylamide gel electrophoresis. c Only six linkage groups have been identified by mutation, although seven are expected based on cytological detection of seven

_ _ ~

tS tS cycA + W A + whiB + b d + mad+ bsgB +

containing media, forcing the cycAl mutation in all isolates.

chromosomes (see NEWELL 1982). The unmarked linkage group is V for historical reasons.

FIGURE 2.-Complementation of Dis" phenotype of motA1552. Samples were prepared from the same cultures used in Figure 1 and separated by SDS polyacrylamide gel electrophoresis. Indi- cated molecular weight standards, in daltons, were: phosphorylase b, 94,000; bovine serum albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; soybean trypsin inhibitor, 20,100; a- lactalbumin, 14,400. Lanes a and h, discoidin I (the major, higher molecular weight band) and discoidin 11; b, MC2, 21"; c, MC2, 27"; d, HU1136,21"; e, HU1136,27"; f, DMCIJO, 21"; g, DMC150, 27".

dependently of markers on linkage groups I, 111, IV, V I and VII, and with acrA' on linkage group 11. These data indicate that the motA locus is on linkage group 11. Segregation data from all motA1552/motA+ diploids examined were consistent with this conclusion.

One segregant class, motAl552whiB513, was not recovered in the cross reported in Table 2. Skewed segregation such as this is common in D . discoideum

parasexual genetics (NEWELL 1982). The absence of such segregants left open the possibility that there was information on linkage group I11 that was necessary but not sufficient for the expression of motA1552 phenotypes. This was ruled out by data from other dip- loids. In DMC149 (MC2 X MC204) the motA' parent carried whiAlacrAltsgD12 on linkage group I1 and bsgA5 on linkage group 111. The bsgA5 linkage group I11 is from a wild type strain, and therefore does not carry axeB1 or any other marker that might pre-exist on the axenic linkage group I11 in MC2. Out of 266 haploid segregants in a particular experiment, 67 should have carried motA1552 based on Whi+ and Acr+ phenotypes. Of these, 24 were reassorted for the linkage group I11 marker, bsgA5, and all 24 were found to be Tsg-. Mot and Dis phenotypes were scored for four Whi' Acr'Tsg-Bsg- segregants, and they were MottSDistS. Therefore, expression of motA1552 pheno- types did not require linkage group I11 from MC2 or its parent, AX3.

To confirm the mapping of moa1552 to linkage group 11, selected mitotic recombinant diploids were examined. To this end, a mutation at acrA on linkage group I1 was selected in MC349, a strain carrying motAI552, by plating on methanol-containing me- dium (WILLIAMS, KESSIN and NEWELL 1974). This haploid, MC445 (which was stable for methanol re- sistance, see below), was fused with TS12, yielding the methanol-sensitive diploid, DMC144. acrA-l acrA- diploids were screened from among derivatives of DMC144 selected for methanol resistance. The three mitotic recombinant diploids identified (at least two of which were independent) were Tsg-MottsDists. Thus, selection for recombination at acrA can simul- taneously generate diploids recombinant at motA, demonstrating that these loci are linked.

A number of motAl552/motA+ diploids were found to yield haploid recombinants between whiA and acrA at unusually high frequency (see below). The three

Analysis of motA1552 429

w h i A 1 a c r A 2 O 1 + t s g D 12

0 I I I I I I 1 I

0 I I I I I I I I

+ + m o t A 1552 + FIGURE 3.--Gene orders on linkage group 11. This placement

of the motA locus is tentative due to uncertainty regarding the generation of the recombinants. The linkage groups I1 of DMC95 are diagrammed and the presumed sites of exchange that gener- ated the recombinant classes described below are indicated. The location of motA with respect to tsgD is arbitrary. Seven haploid recombinants isolated from DMC95 (MC279 X HK12) and one from DMC149 (MC2 X MC204) were analyzed. Both motA+ parents carried three mutations on linkage group 11, at whiA, acrA, and tsgD in order of proximity to the centromere. All phenotypes for linkage group I1 mutations were scored. Three recombinant classes were recovered: whiA+acrA201motAftsgD12 (one isolate), whiAlacra+motAl552tsgD+ (five isolates), and whi- AlacrA+motA+tsgDlZ (two isolates). These data map motA centrom- ere-distal to whiA. If the presumed double recombinants (whi- AlacrA+motA+tsgDlZ) share the precise site of exchange in the whiA-acrA interval with the single recombinants, then motA is also centromere-distal to acrA.

phenotypes of motA1552 were not separated in any of these recombinants. These fortuitous haploid re- combinants allow motA to be ordered with respect to other mutations on linkage group IT (see Figure 3). Such an analysis assumes that these haploids result from classical mitotic recombination (WELKER and WILLIAMS 1982a). However, these recombinants are also consistent with gene conversion at the whiA or acrA locus (depending on the recombinant), as well as multiple exchanges, and thus might not be classical mitotic recombinants. Unusual caution in interpret- ing these data is necessary because of the elevated levels of genetic activity observed in this region (see below), and therefore the gene order in Figure 3 is tentative.

The three mutant phenotypes assigned to mot- A1552 co-segregated in all crosses performed. In addition, they were not separated in the haploid or diploid mitotic recombinants. These data support the conclusion that the Tsg-, Mot" and Dids phenotypes result from the same mutation, motA1552. However, it should be noted that the resolution of mitotic recombination is limited, and that few (if any) of the recombinants analyzed resulted from an exchange close to motA.

Variation in expression of motAI552 phenotypes and temporal separation of motility loss and dis- coidin I production in MC517: The degree of tem- perature sensitivity varied among strains carrying

motA1552. Clonally maintained stocks of MC2 had EOP values at 27" of to The small clones isolated at 27" replated with an EOP of close to 1 at 27" (KAYMAN, REICHEL and CLARKE 1982). A strain isolated as a motA1552 segregant, MC279, had an EOP at 27" of < loF6, and the tiny clones thus isolated were not Tsg' revertants. Despite the failure to select Tsg+ revertants of MC279 at 27", variants of both strains with higher 27" EOP values, often approach- ing 1, were isolated simply by screening small num- bers of clones following long term bulk passaging at 21". This variation in EOP reflected differences in the amount of growth at the restrictive temperature either in addition to or instead of changes in mutation rate, since these variants also had increased growth rates in bacterial suspension culture at 27". Thus, the Tsg- phenotype of motA1552 was not stable.

The amount of time at restrictive temperature required for significant loss of motility and accumu- lation of detectable amounts of discoidin I also dif- fered among motAl552 strains, while being largely reproducible for any given strain. Translocation rates and discoidin I levels were examined at various times following shift of bacterial suspension cultures to restrictive temperature. Movement was determined from time-lapse records of fields of cells, and discoi- din I was monitored by immunofluorescence, a sen- sitive technique that detects very low levels of the protein. The shortest time interval observed between upshift and expression of mutant phenotypes was two to three hours for MC2, the longest was 12-14 hr for MC349.

Variation in the time of expression of the Mot" and DistS phenotypes allowed exploration of their relationship to each other. For one motA1552 segre- gant, MC517, the loss of motility preceded by 3 hr the appearance of discoidin I detectable by immu- nofluorescence. Figure 4 plots motility data and Figure 5 shows discoidin I immunofluorescence for MC5 17, each as a function of time after upshift. Loss of motility, seen both as the sharp drop in average translocation rate and as the appearance of nonmotile cells, occurred by four hours. Discoidin I was not detectable in a significant number of cells before 7 hr. Although we do not have an absolute measure of the minimum amount of discoidin I detectable by the immunofluorescence method, our data indicate that the threshhold is extremely low (CLARKE, KAY-

MAN and RILEY 1987). We therefore believe that discoidin I was detected soon after its synthesis was initiated. These results indicate that the discoidin I synthesized by motA1552 strains is not responsible for their loss of cell motility.

Discoidin I accumulation during axenic growth: During axenic growth of AX3, discoidin I accumu- lates to nearly 1% of cell protein (BISWAS, KAYMAN and CLARKE 1984). This is similar to the maximum

430 S. C. Kayman, R. Birchman and M. Clarke

l 2 r t-

w o Y C

wb 0.4 00

I U 2 4 p g 0.2

0

0 0

0

I+; I I I , I ,

0

O L L 0 2 4 6 8 10 12 14 16

TIME AFTER SHIFT TO RESTRICTIVE TEMPERATURE ( HOURS 1

FIGURE 4.-Loss of motility by MC517. Passaged suspension cultures of MC517 were shifted to 27" and sampled at various times. Motility was assessed from videotape records covering 20 to 30 min; between 15 and 25 cells were monitored for most time points. Each symbol represents a different experiment. Data are plotted as mean translocation rate in the upperpanel, and as fraction of nonmotile cells in the lower panel.

level reached during development (SIU et al. 1976), and is readily detected by SDS gel electrophoresis. However, as shown in Figure 6, axenic derivatives of AX3 carrying a wild type linkage group I1 did not accumulate sufficient discoidin I during axenic growth to be detected on SDS gels. This observation identified a mutation, designated daxA1551, that is required for accumulation of large amounts of dis- coidin I during axenic growth, the Dis"" phenotype. The much more sensitive immunofluorescence tech- nique showed that daxA+ strains did contain a small amount of discoidin I at moderate cell density during axenic growth (M. CLARKE, unpublished observa- tions). This is in contrast to cells growing at low den- sity axenically or on bacteria, which contain no detect- able discoidin I (CLARKE, KAYMAN and RILEY 1987).

DMC105 (MC293 X HU49) was constructed for the genetic analysis of the Dis"" phenotype. MC293

was derived from NP2, a Tsg- derivative of AX3; HU49 was derived from M28 and TS12, genetically marked strains that have no axenic parentage. Se- gregants known to carry the axenic linkage group I11 by standard genetic markers were selected for analysis, because this is the one linkage group re- quired for axenic growth (CLARKE and KAYMAN

1987). The level of discoidin I during axenic growth was monitored by SDS gel electrophoresis; data for typical segregants with high or low levels of discoidin I are presented in Figure 6. (Low discoidin I was defined as not detectable by SDS gel.) Immunoblot analysis (Figure 7) confirmed that the band in Figure 6 that co-migrated with discoidin I and varied be- tween the two classes of segregants was in fact dis- coidin I . Linkage data (Table 3) showed that strains accumulated discoidin I to high levels during axenic growth if and only if they carried linkage group I1 from the axenic parent.

The segregants reported in Table 3 all carried linkage group I from the axenic parent. This was necessary to allow unambiguous identification of the axenic linkage group I11 by a strain's Tsg- pheno- type. However, this left open the possibility that linkage group I also carried information necessary for accumulation of discoidin I during axenic growth. A small number of segregants carrying linkage group I from the nonaxenic parent and linkage group I1 from the axenic parent (identified by Spr- and Acr-, respectively) were therefore screened to identify those able to grow axenically. Two such strains were identified; both had high levels of discoidin I during axenic growth (Figure 6, lane c). Thus, linkage group I from AX3 was not required for expression of the Dis"" phenotype. d d ' strains were found to be capable of normal

accumulation of discoidin I both during development (Figure 6, lane g) and in bacterial culture (S. C. KAYMAN, unpublished observations). Thus, d d 1 5 5 1 is not required to overcome a defect in normal discoidin I induction introduced during isolation of AX3, but rather causes cells to accumulate large amounts of discoidin I protein specifically during axenic growth.

Genetic abnormalities in MC2: MC2 also manifests two types of elevated genetic activity on linkage group 11. The possibility of a relationship between either of these phenomena and motA1552 is unresolved.

First, some motA1552/motAf diploids showed very high recombination (approximately 8%) between the whiA and acrA loci. For DMC95 (MC279 x HK12), seven out of 77 haploid segregants (9%) selected on ben late were recombined in this interval. DMC 11 5 (MC2 x HK12) also showed high frequency recom- bination in this interval: 6% (8 of 128) among haploids selected on cycloheximide and 9% (12 of 133) among haploids selected on ben late. Five of the 20 recom-

43 1

0 4.5 7.5 9 13 15 FIGURE 5.-Presence of discoidin I in MC517. Samples from the experimental culture plotted as (M) in Figure 4 were prepared

for indirect immunofluorescence at the indicated times (in hours). They were stained with rabbit anti-discoidin I antiserum followed by fluorescein-conjugated anti-rabbit IgG. Immunofluorescence and phase images of each field are presented (magnification, X 220; bar = 25 pm).

FIGURE 6.-Discoidin I accumulation during axenic growth. Samples b-f were prepared from passaged axenic cultures at 1-3 X IO6 cells per ml; sample g was prepared from bacterially grown cells allowed to develop in suspension for 9 hr. Protein from 3 X lo5 cells was loaded per lane and separated by SDS polyacrylamide gel electrophoresis. Standards were as in Figure 2. a and h, discoidin I (the major, higher molecular weight band) and discoidin 11; b, MC293 (Disax parent of segregants); c, MC347 (Dis"" segregant); d, MC338 (Dis"" segregant); e, MC335 (Dis+ segregant); f, MC329 (Dis+ segregant); g, MC345 (Dis+ segregant).

binants from this diploid were whiA+acrA201. Thus, the high frequency recombination did not depend upon the use of ben late, a drug that induces hap- loidization (WILLIAMS and BARRAND 1978) and prob- ably disrupts the mitotic spindle (WELKER and WIL- LIAMS 1982b), and both classes of recombinant were recovered as a reasonable fraction of the recombinants.

Mitotic recombination is usually observed as dip- loids homozygous for a recessive resistance marker when a heterozygous diploid is selected on the ap- propriate medium. Since the level of total resistant segregants in such situations is normally 10-9-10-4, these diploids that yielded high levels of haploid recombinants might be expected to have abnormally high EOP values on methanol-containing medium (which selects for homozygosity of acrA - alleles). However, in many platings, these and related diploids had normal EOP values on methanol. Either gener- ation of the recombinant haploids is linked to the haploidization process, or the recombination event is constrained such that the homozygous acrA- product is not formed.

Interestingly, a number of the whiAlacrA+ recom- binants from DMC95 were initially scored as Whi+Acr+ haploids. The Whi phenotype was there- fore carefully monitored during the initial processing of segregants from the second cross. Two of the 15 whiA1 acrA + recombinants isolated from DMC 1 15

432 S. C . Kayman, R. Birchman and M. Clarke

TABLE 3

Mapping of darrA15.51, the mutation responsible for axenic accumulation of discoidin I, in DMClO5

Linkage group

Strains or phenotypes I I 1 111 IV VI VI1

MC293 H U49

~~~~~

sprA + LsgE + ncrA1551 LsgA 1 bwnA + nyB15.51" cobA +

sprA 1 Q-E 13 acrA + lsgA + b u d 1 nysB + cobA358

+ - - + - + + - - + + - High discoidin I 8 0 8 0 8 0 6 2 2 6 6 2 Low discoidin I 10 0 0 10 I O 0 6 4 7 3 7 3

Only markers scored in this cross are indicated. See Table 1 for complete genotypes. Segregants were selected on medium containing ben late. Those carrying linkage group I from HU49, determined by round spore shape (Spr- ), were excluded to allow scoring of tsgA1. The linkage group carrying 1sgAl is required for axenic growth.

The nv5B locus has recently been mapped to linkage group VI (WELKER 1986).

a b C d

FIGURE 7.-Identification of axenic discoidin I by immunoblot. Aliquots of the same samples shown in Figure 6 were separated by SDS polyacrylamide gel electrophoresis on a minigel apparatus, transferred to nitrocellulose, and visualized with rabbit anti-dis- coidin I antiserum followed by peroxidase-conjugated anti-rabbit IgG. Relative loadings were as in Figure 6, although absolute amounts were less, due to the different gel systems. a, discoidin I and discoidin 11; b, MC293 (Dis"" parent); c, MC347 (Dis'" segregant); d, MC335 (Dis+ segregant).

were derivatives of Whi+ segregants that continued to generate Whi- derivatives through sequential clonings. Such behavior is consistent with a tandem duplication (WELKER, METZ and WILLIAMS 1982) or a chromosome fragment (WILLIAMS, ROBSON and WELKER 1980) covering the whiA locus.

Preliminary attempts to assign this recombinogenic behavior to either the motA1552 (MC2 and MC279) strains or HK12 yielded complex results. MC2 showed both high (8%, 9 of 119 segregants of DMC173 (MC2 x HU25)) and low (0.3%, 1 of 373 segregants of DMC149 (MC2 x MC204)) recombi- nation between whiA and acrA in diploids with other strains, while the one other diploid of HK12 exam- ined showed an intermediate level of recombination

TABLE 4

Analysis of methanol-resistant derivatives

Instability of methanol resistanceb Mutation rate to methanol

resistance (EOP on Fraction of resistant methanokontaining isolates yielding Degree of

Strain medium, X 10')" sensitive derivatives instability

MC2 1.4 t 0.85 (17) 1115 0. I 8 MC513' 2.6 t 2.3 (17) 0110 MC487 870 2 1600 ( 9) 1 14 0.3 1 MC493d 210 t 170 ( 4) 214 0.22, 0.38 MC3 1.2 t 1.1 (16) 2/16 0.01,0.01

NP2 3.4 t 1.0 ( 7) 1 I4 0.01 22 ( 1)' 014

Means and standard deviation, with number of determinations in arentheses.

'To screen for instability of methanol resistance, independent resistant isolates were cloned twice on methanol-containing me- dium and then bulk passaged twice on nonselective medium. Approximately 100 clones from nonselective medium were then tested for methanol resistance. For each isolate that yielded sensitive derivatives, the fraction of the clones from nonselective medium that were methanol-sensitive is shown as "degree of instability."

MC513 is a methanol-sensitive derivative of MC514, the un- stable methanol-resistant derivative of MC2.

MC493 is a methanol-sensitive derivative of the unstable methanol-resistant derivative of MC487.

'This unusually high EOP probably represents a 'tjackpot" event; the four clones examined probably carry the same mutation.

(4%, 7 of 161 segregants of DMC157 (HK12 x NP2)). This was surprising, since other groups have not seen elevated recombination when working with HK12 (KASBEKAR, MADIGAN and KATZ 1983; WELKER 1986). Crosses giving extremely high recombination were not restricted to a specific batch of media compo- nents, ruling out certain possible artifacts. Thus, although the extremely high recombination was only seen in crosses with MC2 background, another factor, possibly from the axenic genetic background, must also be involved (see DISCUSSION).

The other genetic abnormality seen in MC2 in- volved the stability of mutations at the acrA locus. When D . discoideum strains are plated on medium

Analysis of motAI552

TABLE 5

complementation d y s i s of methanol resistance in MC514, the unstable derivative of MC4

433

Diploid

DMC 176 DMC 177 DMC 174 DMC 178 DMC184 DMC185 DMC186 DMC 187

Haploid fused to MC5 14

MC288 MC288 HU25 HU25 XP55 XP55 TS12 TS12

Presumed wA" genotype as constructed

acrA-lacrA- acrA-lacrA- a o A - IacrA- acrA - IacrA- acrA-lacrA+ M A - lacrA+ acrA- lacrA+ acrA- lacrA+

containing medium and implied WA genotypeb No. of clones with indicated EOP on methanol

>0.5 MA- IwA- UTA - IwA + MTA+ ILUTA+

10-3- 10-5 4 0 - 5

1" 2

2 2 4 4

2 2

This assumes MC514 carries an acrA allele. Because typical UTA mutations confer recessive resistance to methanol, a diploid homozygous for an acrA- allele grows in the presence

of methanol with an EOP near 1. A diploid heterozygous for an u r A - allele cannot grow in the presence of methanol; haploid segregants carrying the acrA- allele and diploid recombinants that have become homozygous for it do grow, typically within the indicated range of EOP. A diploid homozygous for m A + requires a mutation followed by haploidization or recombination to yield growth on methanol, and therefore has an extremely low EOP. Thus, the data for DMC174, DMC178, DMC186 and DMC187 are unusual (see text).

The second clone of this diploid examined had an anomalous EOP of 0.07.

containing 2-2.5% methanol, resistant mutants are isolated at frequencies near the mutations conferring methanol resistance have been mapped to acrA (WILLIAMS, KESSIN and NEWELL 1974). We are not aware of selection for methanol resistance yielding mutations at loci other than acrA, nor of any reports of unstable acrA mutations. However, 1 of 15 independent methanol-resistant derivatives of MC2, MC514, was extremely unstable. During bulk passag- ing on nonselective medium, methanol-sensitive de- rivatives accumulated (Table 4). A number of other strains with MC2 background, apparent simple se- gregants as well as recombinants in the whiA-acrA interval, yielded similarly unstable methanol-resistant derivatives.

This instability probably represents reversion of a mutation responsible for the methanol resistance, because the two methanol-sensitive derivatives ex- amined (MC5 13 and MC493) did not differ from their methanol-sensitive "grandparents" in mutation frequency or instability for methanol resistance (Table 4). Complementation analysis indicated that MC514 does carry an acrA allele (Table 5) . Note that this analysis was complicated by a very high level of instability (presumed reversion of the acrAl552 mu- tation in MC5 14) in certain diploids (those made with HU25 and TS12).

In general, the mutation frequencies to methanol resistance were normal in strains that yielded highly unstable acrA mutations. The one exception was MC487, a whiAlacrA+motA'tsgDl2 double recombi- nant isolated from a diploid carrying motA1552. This strain had a very high mutation frequency to meth- anol resistance (Table 4). This was not a general property of double recombinants, since the mutation

frequency to methanol resistance of a second such recombinant was normal ( S . C. KAYMAN, unpublished observations).

Methanol-resistant derivatives of MC3, another motility mutant selected from AX3 at the same time as MC2, and NP2, an unrelated Tsg- derivative of AX3, were also examined. Surprisingly, these strains were similar to MC2 in their fraction of unstable derivatives, but the degree of instability was much less than that of strains derived from MC2 (Table 4). At the level of screening thus far carried out, we do not know whether all acrA mutations isolated in axenic strains are unstable at a level of 0.001-0.002 in our procedure, or if an occasional mutation is unstable at 0.005-0.02. If the latter, the unstable mutations in MC2-related strains may be similar in nature to those in other axenic strains, and the difference may be in the level of an activity that mediates the rever- sion to methanol sensitivity.

These observations argue that the extreme insta- bility of occasional awA mutations is the phenotype of a mutation that arose during the isolation of MC2. Available data on segregants are not sufficient to determine whether this mutation is linked to mod, due to the statistical nature of the assay for the mutation. However, if motA1552 is itself responsible for the increased instability at acrA, then generation of the complex recombinant MC487 must have cre- ated a variant at motA with acrA-related activities but lacking the established motA1552 phenotypes.

DISCUSSION

Linkage data presented here support the conclu- sion that the Mot-, Dids, and Tsg- phenotypes of

434 S . C. Kayman, R. Birchman and M. Clarke

MC2 are all phenotypes of a single mutation, mot- A1552. Our finding that motility loss can precede the appearance of discoidin I protein by at least three hours in motA1552 strains argues strongly that, con- trary to the suggestion of SPRINGER, COOPER and BARONDES (1984), the motility defect is not mediated by discoidin I. It is also unlikely that discoidin I synthesis is induced as a consequence of the loss of motility, since a number of other motility-defective mutants do not produce discoidin I (BISWAS, KAYMAN and CLARKE 1984). It is more likely that the Motts, DiP, and Tsg- phenotypes are pleiotropic effects of motA 1552.

The linkage group I1 of AX3, on which motA1552 was isolated, was shown to carry a mutation that affects discoidin I accumulation. This mutation, daxA1551, is responsible for the accumulation of large amounts of discoidin I by AX3 cells during axenic growth.

The basis of the effect of dajcA on axenic discoidin levels remains obscure. The density-dependent reg- ulation of discoidin I and other early developmental proteins appears to function normally during axenic growth of AX3 (CLARKE, KAYMAN and RILEY 1987). This fits well with the suggestion that the presence of early developmental proteins during axenic growth is due to normal induction of an early segment of the developmental program that does not require starvation (BURNS, LIVI and DIMOND 1981). It was therefore surprising to find that this behavior re- quires the daxA1551 mutation, at least for discoidin I. The phenotype of daxA + strains is a low level of discoidin I at moderate cell densities during axenic growth, which is distinct from both the absence of discoidin I typical of low density cells from bacterial or axenic culture, and the higher levels seen during development, at moderate cell densities during axenic growth of daxA- strains, and in higher density cells from bacterial culture. This low level of discoidin I might result from a lower synthetic rate, or from loss by secretion or proteolytic degradation. The major reorganization of the endocytotic machinery of the cell that occurs during adaptation to axenic conditions (KAYMAN and CLARKE 1983; CLARKE and KAYMAN 1987), and the complex patterns of secretion that are seen during axenic growth of AX3 (BURNS, LIVI and DIMOND 1981), suggest that the latter possibilities are plausible. Otherwise, an additional level of regulation of discoidin I synthesis is implied. Additional work is needed to determine the level of daxA1.551 action and its effect on other early developmental proteins.

The presence of duxA1551 complicates attempts to understand the action of motA1552. Other mutations have also been mapped to the linkage group I1 of AX3 (GINGOLD and ASHWORTH 1974; NORTH and WILLIAMS 1978; WILLIAMS, KESSIN and NEWELL 1974). To understand motA1552 action, it is important to

know if it is allelic to any of the preexistent mutations on the axenic linkage group 11, particularly daxA1551. In addition, it is important to know if the expression of any or all of the motA1552 phenotypes is dependent on one of these other mutations. Unfortunately, the disparity among the phenotypes of these mutations and their presence in motA1552 haploids suggest that complementation analysis will not be informative.

During the course of this work, several types of genetic instability were found to be associated with MC2. Expression of the motA1552 phenotypes was variable. This was true among different segregants carrying the mutation, as well as among different clones of individual motA1552 strains following bulk passaging. Although the segregant-to-segregant variation may reflect preexistent differences in ge- netic background, the clone-to-clone variation must result from some form of instability directly or indi- rectly affecting the motA locus or its products.

Two other types of instability were traced to MC2. As far as we know, neither directly involves genes affecting discoidin I, although a relationship to motA1552 has not been ruled out. The action of the presumed mutation(s) responsible for these instabil- ities cannot be simple, and something in the axenic genetic background may be involved.

One of these instabilities involved mutations at acrA, which confer resistance to methanol. While MC2 and many of its derivatives yielded a small fraction of methanol-resistant derivatives that were highly unstable, other axenic strains yielded a similar fraction of methanol-resistant derivatives that showed a lesser degree of instability.

Second, recombination between whiA and acrA on linkage group I1 was extremely high in some diploids carrying MC2 or a related strain. A survey of the literature suggests that normal recombination rates between acrA and whiA are less than 1 % (e.g., NEWELL et al., 1977; RATNER and NEWELL 1978; WELKER and WILLIAMS 198213). However, there are two published crosses (GINGOLD and ASHWORTH 1974; ROTHMAN and ALEXANDER 1975) and one reported here (which did not involve the MC2 background) that showed somewhat elevated recombination (approximately 3%) in this interval. This degree of elevation of recombination has been reported as the result of the presence of translocations (WELKER and WILLIAMS 1985). Diploids containing MC2-derived strains showed either the extremely high level (approxi- mately 8%) or the normal level (approximately 0.3%) of recombination, depending on the other haploid parent, indicating that another factor is required. All diploids that yielded recombinants in this interval at greater than 1%, both reported here and in the literature, were between strains heterozygous for axenic genetic background, suggesting that this factor is in the axenic background.

Analysis of motAl552 435

The involvement of unstable Whi+ intermediates in the generation of at least some of the high fre- quency recombinants isolated here is reminiscent of a previous report of similar segregants. They were derived from parasexual diploids formed between haploids of opposite sexual cycle mating types. Par- asexual crosses between strains of opposite mating type display vegetative incompatibility, yielding dip- loids at less than 1% of the frequency of crosses between strains of the same mating type (ROBSON and WILLIAMS 1979). Such diploids have become homozygous for the mating type locus and the de- terminants of vegetative incompatibility on linkage group I. Some diploids of this type were found to generate unstable Whi+ segregants that carried a partial linkage group I1 with an apparent endpoint in the whiA-acrA interval (WILLIAMS, ROBSON and WELKER 1980; WELKER et al.1986). The formation of the chromosome fragment probably involved recom- bination. The most likely causes of these unstable Whi' segregants were an unusual translocation or functions involved in establishing vegetative incompatibility.

Many of the behaviors that we observed are similar to those of mutations that result from insertion of mobile genetic elements in other systems. High re- version frequencies (FEDOROFF 1983; LIM 1979; JACK 1985) and transitions among multiple phenotypic states by rearrangements linked to the insertion (COEN, CARPENTER and MARTIN 1986) or by action of unlinked effector loci (WINSTON et al. 1984) have been seen. It is of interest that the yeast mating type locus has such effects on T y insertions (ERREDE et al. 1980). P elements, which are responsible for one of the hybrid dysgenesis systems of Drosophila, cause elevated premeiotic recombination (SINCLAIR and GREEN 1979) and chromosome breakage (HENDER- SON, WOODRUFF and THOMPSON 1978). The similarity of these observations to our findings suggests that mobile elements may also underlie our results.

Instabilities that may be analogous to those pre- sented here have been reported for strains carrying other linkage group I1 developmental regulatory mutations, disA50 (ALEXANDER, SHINNICK and LERNER 1983), and fstAl (SOLL et al. 1987). Furthermore, POOLE and FIRTEL (1 984) have reported chromosome rearrangements in AX3 closely linked to the discoidin I structural genes, which are on linkage group I1 (D. WELKER, personal communication). These rearrange- ments involved a pair of putative transposons.

It is intriguing to speculate that these various instabilities are related. The activity of mobile genetic elements, perhaps a system involved in establishing vegetative incompatibility via a hybrid dysgenesis-like mechanism, might be responsible. Alternatively, aber- rant functioning of a developmental regulatory sys- tem with a recombinational mechanism (e.g. inversion

(BUKHARI and AMBROSIO 1978; ZIEG, HILMEN and SIMON 1978) or transposition (HERSKOWITZ 1983; BORST 1983)) might provide the explanation. Further work is necessary to explore these possibilities.

We are grateful to SAMUEL BARONDES for generously supplying the discoidin-I antiserum used in this study. This work was supported by grants from the National Institutes of Health to M.C. (GMll301 and GM29723).

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Communicating editor: S. L. ALLEN