Mutants of Polysphondylium pallidum altered in cell aggregation and in the expression of

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The EMBO Journal vol.4 no. 10 pp.2525 -2532, 1985

Mutants of Polysphondylium pallidum altered in cell aggregation and in the expression of

a

carbohydrate epitope

on cell

surface glycoproteins

D.Francis1, K.Toda, R.Merkl, T.Hatfield1 and G.Gerisch

Max-Planck-Institut fur Biochemie, D-8033 Martinsried beiMunchen, FRG, and 'School of Lifeand Health Sciences, University of Delaware, Newark, DE 19716, USA

Communicatedby G.Gerisch

Mutants ofthecellularslime moldPolysphondyliumpallidum have been selected using a cell sorter and a fluorescent- labeledmonoclonalantibody,mAb293. This antibody blocks cell adhesion when applied asFab, and recognizesacarbo- hydrateepitopecontainingL-fucose.This epitope is expressed onthe cellsurface and is present on >10membraneglyco- proteins ofdifferentapparentmol. wts.

Twenty

mutants were obtained which didnotbind mAb293 when tested at 2 h of starvation. After longer periods of starvation the epitope became detectable in themutants. Inallthese mutants aggre- gationpatterns wereatypical. Generally streams ofcellsthat were radially orientated around aggregation centers were missing or were much shorter than in wild-type. Genetic analysisdemonstrated that aberrant aggregationwaslinked tothealterationin carbohydrate epitope expression. One mutant wasunstableand gaverisetosubclonesinwhich almost no antibody binding wasobserved, even after 24 h ofstarvation, andonly fewaggregationcenterswithno streams or veryshort ones wereformed.Theseresultsindicate that the capability ofthecellstoaggregateis correlatedwith theex- posure ontheirsurfaces ofthecarbohydrateepitoperecogniz- edby mAb 293, whose functionin development remainsto be established.

Keywords:fucosylation/contactsites/cellsorting/lectins/cellular slime molds

body, mAb 293, raised against this pair ofglycoproteins completely blocked adhesion of suspended P.pallidum cells and was neutralized by the purified carbohydrates (Toda etal., 1984a).

These observations prompted us toselect mutants that aredefec- tive in the carbohydrate epitope recognized by mAb 293.

Inimmunoblots mAb 293 recognized not only the 64-kd pair but also between 10 and 20 otherglycoproteins of lower and higher mol. wts. Binding of the antibody to all these glycoproteins was completely blocked by free L-fucose (Toda et al., 1984b). This result does not mean that the antibody recognized only L-fucose residues, but indicates that the affinity of the anti-

Mutagenization and growth of the cells for 2 to 3 generations

V V

Labeling with mAb 293 plus FITC-conjugated anti-mouse IgG

V V

Sorting in a FACS IVcell sorter

Light scattering IFluorescence

Cloning onto LP agar plates

Introduction

Polysphondylium pallidum is a cellular slime mold, like Dic- tyosteliumdiscoideum, whose cells aggregate after ^- 7 h of starvation, forming large streams radially orientated around aggregationcenters. The system responsible foraggregationof P. pallidum cells differs from that of D. discoideum such that cells of thetwospeciessort outinto separate aggregatesnot only because they respond to different chemoattractants(Shimomura et al., 1982), but also because ofspecies-specific membrane- membrane recognition (Gerisch et al., 1980).

Cell surface glycoproteins and their participation incell adhesion have been studied in P.pallidum (Bozzaro andGerisch, 1978; Steinemannetal., 1979;Bozzaroetal., 1981)inparallel to similar work onD. discoideum (Mullerand Gerisch, 1978;

Murray etal., 1981, 1983; Ochiai etal., 1982; Yoshida et al., 1984;Bertholdtetal., 1985). The adhesionblockingactivityof polyclonalFabfrom antiseraraisedagainstwhole membrane frac- tions ofaggregation-competent P.

pallidun

cells was neutralized to 80% by a purified carbohydrate fraction (Toda etal., 1984a).

Thecarbohydrate was obtained by hydrazinolysis of two glycoproteins

that

form a double band in the 64-kd region after SDS- polyacrylamide gel electrophoresis. Fab ofa monoclonal anti-

Blotting of colonies onto nitrocellulose andlabeling with 125I-mAb293

V V

Subculture of cells from unlabeled colonies

< l \~~~

SDS-polyacrylamide gel electrophoresis and immunoblotting

with mAb 293

assay of cell aggregation

on LPagar plates

genetic analysis to demonstrate linkage ofcarbohydrate defect and defectiveaggregation Fig. 1. Experimental protocolforthe selectionand analysisof mutants defectivein ep293expression. In mostof theexperiments, cell-sorter selection of the cellswas repeated after severalgenerations ofgrowthbefore the cells wereclonedontolactose-peptone (LP)agar. The vertical white lines inthephotograph show the boundaries of the windows fromwhich cellswereselected. Intensity oflight scatteringasa measure of cell size, and of fluorescenceareplottedontheabscissa, relative numbers of cellson theordinate. The window of fluorescencecorrespondstothe

autofluorescence intensity ofthe cells. Thephotograph wastakenduringa secondrunofselectionwhere unlabeled cellswerealreadyenriched.

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o0

40

Fig. 2.Colony blot labeled with[125I]mAb293. Approximatesizes of coloniesatunlabeledpositionsareindicated. The two larger unlabeled colonieswere theorigins oftwomutants; the three smaller coloniesprovedtobewild-type; they were too young to bedetectably labeled.

Table1. Yield ofmutantsafter one ortworunsofsortinginaFACS IV cell sorter

Experimentno. 1 2 3 4

Mutagen U.V. MNNG U.V. MNNG

Presorted Yes No Yes Yes

Ratio of sortedtototal cells 3.4 x 10-3 2.4 x 10-3 5.7 x 10-3 1.1 X 10-2

Ratio ofgrowingtosortedcells 0.47 0.30 0.19 0.65

No. ofclones examined 89 85 108 124

No. ofstablemutants 5 2 6 7

Designationofmutants PN6002; 6003; 6004;6005; PN6007; 6008 PN6009; 6010; 6011; 6012; PN6015; 6016; 6017; 6018;

6006 6013; 6014 6019;6020; 6021

bodytoL-fucosewashigh enoughto-preventitsbindingtothe complete carbohydrate chains, where it recognizes an epitope thatincludesnotonlyL-fucose butalso othersugars.Methylation analysis revealed that L-fucosewasalwaysinaterminal

position

at thenon-reducing ends of the

protein-linked oligosaccharide

chains(Todaet

al.,

1984b). Therefore, mAb 293 could be used toselect, inacell sorter, mutantsinwhich either theexpression of thewholesetofglycoproteins recognized by this antibody is defective,orinwhichsynthesis, transfertoproteins,orprocess- ing of the carbohydrate residues is blocked.

InP.

pallidum

sexual developmentcanbeinducedby co-culture oftwohaploidstrainswithopposite matingtypes(Francis, 1980).

Undertheseconditionsmacrocysts areformed.These arethick- walledaggregates ofcells inwhichzygoteformation and meiosis is carriedout.Thussexual recombinationcanbeusedin P.palli- dumforlinkage analysisof mutants(Francisand Rupar, 1983).

In the present paper we report on the selection and genetic analysisof 20mutantstrainsinwhich delayed expressionof the

carbohydrate epitope recognized by

mAb293wasassociated with

aberrations

inthe

pattern

ofcell

aggregation.

This

epitope

will be designated ep 293.

Results

Selection

of

mutants

Aftermutagenesiswithnitrosoguanidine (MNNG) or ultraviolet

irradiation

(u.v.),cells ofstrain PN600weregrown onbacteria for 2-4 generations, thenwashed and shaken

in

non-nutrient buffer, and labeled after 2 h of starvation with mAb 293 and FITC-conjugated sheep anti-mouse IgG. Unlabeled cells were selectedbyuseofaFACS IVcellsorter.They were either direct- lycloned on agarplates usingasingle-celldeposition system in combination with the cell sorter, or wereregrown onbacteria, starved, labeled with antibodies and sorted fora second time.

The clonedcells wereallowed to grow into colonies of 0.5-1 cm diameter which contained 1-2 x 105cells (Figure 1). Position and size of thecolonies formed were marked on the bottom of the plates before the colonies were blotted onto nitrocellulose

Aik":

.11.t,

.'n

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Polysphondylium carbohydratemutants

TableII. Phenotypic characteristics of wild-type PN600 and mutant strains with defective ep 293 expression

Strain Generation Aggregation Other characteristics time

PN600 3.3 Normal

PN6002 4.0 Atypical

PN6003 n.d. Atypical Macrocysts not germinating

PN6004 n.d. Atypical -

PN6007 3.8 Atypical -

PN6016 n.d. Atypical No macrocysts

PN6017 3.3 Strongly reduced Numerous microcysts; no macrocysts

PN60181 3.2 Strongly suppressed Derived from unstable mutant PN6018

PN60183 3.1 Strongly suppressed Derived from unstable mutant PN6018

PN6019 3.8 Atypical

PN6020 4.0 Atypical No macrocysts

PN6021 3.2 Atypical No macrocysts

filters,labeled with

[1251]mAb

293 andautoradiographed (Figure 2). Serial dilutionofwild-type cells showed that 14cells gave a noticeably weaker signal than 105 cells, and 103 cells could barelybe detected. Thisindicatesthatclones of mutant cells which produce one tenth or less of the normal amount of antibody- binding sites could be easily distinguished from wild-type.

Afterblotting, few cells remained onthe original plates, but these were enough for starting new cultures from clones that showedweak ornoexpressionof ep293onthe blots. Cells from eachclone of interest wererecloned, onesubclone grown up in suspension culture,and itscellsharvested after 2 h of starvation.

Glycoproteinswerelabeled withmAb293 after

SDS-polyacryl-

amidegelelectrophoresis of total cellularproteins and

blotting

ontonitrocellulose filters. Infourindependent mutagenesisexper- imentsatotal of406 cloneswerescreened,from which 20proved tobemutantstrains that didnot

significantly

express ep293 after 2 h of starvation (Table I). At least six mutants werethe out- comeof

independent mutagenic

events,sinceweobtainedmutants in each ofthefour experiments, and in the fourth experiment twomutants, PN6017 and

PN6018,

were

distinguishable

from the others as reported in the next section.

Phenotypic characteristics

of

the mutants

With theexceptionofPN6018allmutants grewwellinsuspen- sion cultures with EscherichiacoliB/ras afood bacterium. Gen- erationtimesweredetermined for nine of themutantsand found to be close to 3.3 h, the generation time of

wild-type

PN600 (Table

II).

This resultindicatesthat ep293, which was not ex-

pressedduring the growthphaseinthe mutants,wasnot

import-

antfortheuptakeofbacteriaorforcell division. PN6018 grew veryslowlyinsuspension. Becausethismutant wasunstable and gaverisetofaster

growing subclones,

its

generation

timecould notexactly bedetermined. Two ofitssubclones, PN60181 and PN60183, were selected and included in Table II.

Shape of both aggregates and fruiting bodies was estimated onlactose-peptoneagar with E. coli B/r as food bacterium. Ag- gregationinall mutants differed fromthat in wild-type.Aggre- gation designated 'atypical' in Table II was characterized as follows. The aggregates often had no streams, or hada wheel- like appearance due to the formation of stubby streams ofuniform length instead of the long branching streams seen during aggre- gationofthewild-type (Figure3).Eventhoughasingleaggregate of a mutant may be like wild-type in appearance, the collection of aggregates on a plate allowedunequivocal recognition of the mutants. Many of the mutant amoebae remained as a smooth lawn between theaggregates, and the fruiting bodies formed were often smaller and had fewer branches than those of the wild-type.

Two mutant strains were clearly distinguished from the majority of mutants (Table H). One of them, PN6017,extensively produced microcysts after starvation; i.e., most of the amoeboid cellsof the mutant were converted into roundedcells that were coveredby a wall. Only the few remaining cells aggregated and formed fruiting bodies on agar plates. The early conversion of cells intomicrocysts appears to be the reason whyPN6017show- ed strongly reduced aggregation. The second mutant was the slowlygrowing PN6018 which showed atypical aggregates similar to the majority of mutants. However, two stable fast growing subclones of that mutant, PN60181 and PN60183, only sporadically formed aggregates on agarplates; most of the cells remainedunaggregated. Stream formation around the centers was strongly suppressed in these subclones.

Linkage ofatypical aggregation to the ep293defectasrevealed by meiotic recombination

For genetic recombination, all mutants were co-cultured with PN100, awild-type strain of oppositematingtype. Macrocysts, the structures in which zygotes areformed and meiosis proceeds, were obtained from 16 crosses (TableII). Macrocysts from 15 of these crossesgerminated, andthemorphologyof the progeny wasdetermined on lactose-peptone agar with E. coliB/r (Table Ill). The unstable mutant PN6018 gave rise tothree classes of progeny: those with normal,atypical and no aggregation; therefore it has not beenincluded inTable III. Intheremaining 14 crosseswhich weresuccessful,progenywith normalandatypical aggregation wereobtained. Alargeamountof progeny, 910 out of1018, werewild-typeintheir aggregation characteristicsand, with theexceptionof one progeny strainfromPN6002, also in the shape of their fruiting bodies. Thisresult suggests that the mutations determining theaggregation behavior were ofnegative selectivevaluewith respecttosurvivalorhatchingof thehaploid cells that had been formed by meiosis in the macrocysts.

That the mutant progeny weregenuinely haploid products of a zygote rather than leftover parental cells was determinedby testing anumber ofthem formatingtype. Approximately half were ofmating type opposite to the mutant parent. This result is expected sincemating type acts asifcontrolledbytwoalleles of one gene (Francis, 1980).

Progeny of four crosses wereprobed in immunoblotsto see whether ep 293 expression followed morphologicalappearance (Table

Ill).

Allof the 21 progeny from thesecrosseswhichwere classified as mutant on the basis ofmorphology failed to react with mAb 293 at 2 h ofdevelopment, whereas 14 progeny of wild-type morphology did react. Figure 4 shows these results with progeny ofmutantPN6002. Progenyno.

5,

whichwasnot included in Figure 4, showed normal

aggregation

and ep 293 expressionbuthad

fruiting

bodies with curledstalks. Such

fruiting

bodies were not observed in any of the other strains and were

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D.Francis et al.

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Fig. 3.Aggregation and fruiting body formation of mutant PN6002 (A-D) andwild-type PN600 (E- G). PN6002exemplifies the developmental defects seenon LPagar inmutants whose aggregation was designated as atypical in TableII.A,E, sectors from colonies with the growth zone on top, aggregates below this zone, and fruiting bodies on the bottom. B,C,F, aggregates. Those of the mutant usually lacked streams, as shown in B. Exceptionally streams wereformed, as they are in theaggregates in C. But these streams remained much smaller than in typical wild-type aggregates, as they are shown in F.

D,G,fruiting bodies. The scale in E belongs to A and E; that in B to the other parts of the figure.

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Polysphondylium carbohydratemutants

probably the outcome of a spontaneous mutation. Weconclude that in each of the four mutantsthe atypical aggregationis due to mutation ofthe same gene that causes the defect in ep 293 expression. We have not yet performed crosses to determine whether the samegene is mutatedin all four strains, atleast three of which were derived from independent mutagenic events as shown in Table I.

TableIII. Aggregation and ep 293 expression in meiotic recombinants Mutant Aggregation of progeny ep 293 expression in progeny number Normal Atypical Withnormal aggr. With atypical aggr.

PN6002 143 6 7/7 0/5

PN6004 26 0 n.d. -

PN6005 63 16 n.d. n.d.

PN6006 58 19 n.d. n.d.

PN6007 55 13 3/3 0/5

PN6008 52 13 n.d. n.d.

PN6009 121 4 n.d. 0/4

PN6010 65 5 n.d. n.d.

PN6011 66 25 4/4 0/7

PN6012 49 1 n.d. n.d.

PN6013 77 2 n.d. n.d.

PN6014 65 3 n.d. n.d.

PN6015 47 0 n.d.

PN6019 23 1 n.d. n.d.

wt

Delayed expression ofep 293 in the majority ofmutants Since our mutants were selected at 2 h ofstarvation, it remained open whetherthey were absolutely devoid of ep 293, or whether expressionofthisepitopewasonly delayedtooccur atlaterstages ofdevelopment. Toclarifythispoint,all mutants werecultivated in suspension in order to assay them by immunoblotting for expressionofep 293 after 22-24 h of starvation. All mutants withatypicalaggregation, asdefinedabove,expressedtheepitope after that period.

Inordertotestwhether themutantantigens recognizedin the immunoblots wereexposed on the cell surface, cells of mutant PN6002 were labeled at0, 6 and 24 h of starvation with mAb 293 and FITC-conjugated sheep anti-mouse IgG. Fluorescence intensity was compared with that of labeled wild-type PN600 cells in aFACS IV cell sorter (Figure 5). While wild-type cells were already strongly labeled at 0 h, labeling of the mutant cells was negligible at this stage. After 6 h of starvation a fraction of the mutant cells waslabeled, and after 24,h substantiallabeling of all cells was obtained.

Continually suppressedep 293 instrongly aggregation-deficient strains

Asreportedearlier in this paper, aggregation was strongly sup- pressedin twosubclones, PN60181 andPN60183, oftheunstable mutantPN6018. To test whether this aggregation deficiency is paralleled by suppression of ep293,both subcloneswereculti- vated insuspension and samples of starved cells werecollected during a period of 24 h. As shown for PN60183 in Figure 6,

N N D D N N D N D D N

Fig.4. Progeny withnormal and defective ep293expressionfrom crossesofmutant PN6002 withPNIOO, awild-type strain ofoppositemating type. Cells were harvestedat2 hofstarvation, andtotalproteinsfrom 1 x 106cells of each strain weresubjected toSDS-polyacrylamide gel electrophoresis, blotting and labelingwith [1251]mAb 293 followedby autoradiography. Wt, wild-typePN600. 1-12, numbers ofprogenystrains (no. 5 has not been includedfor

reasonsgivenin thetext). N, strains developing normally on LPagarplates. D, strains withatypical aggregationand defectivefruiting body formation.

1 2 3 4 6 7 8 9 10 11 12

N

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ii.,

MI!:.-:: :7Rf-

.:.:: i..

.';"k.4 ,:ii: -..i-.:.%.%:

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11 '. re.....11, -, .;

Vv , .,v ^il -.%_I

.t PN60O.

L t'ht ^(e:&IFt;

Oh

6h

24h

FlL zesce n ce

Fig. 5. Cellsorter scansindicating delayed appearance of ep293onthe surfaceofmutantPN6002 cells. Wild-type (leftpanel)andmutant(rightpanel) cells were washedfree ofremainingbacteria atthelategrowthphase, andeither usedimmediately (Oh)oraftershakinginphosphatebuffer for 6or24h.

Atthe times indicated the cellswere labeled with mAb 293 followedbyFITC-conjugated anti-mouseIgG,andanalysed by ^useofaFACS IVcellsorter as indicated in Figure 1.

therewas almostno labeling with mAb 293 detected in either strain even in the latest

samples

taken.

Discussion

Wehaveusedafluorescence-activated cellsorterforthe enrich- mentandcloningof mutants defective in theexpressionofa carbohydrate epitope, ep 293, oncell surface

glycoproteins

of P.

pallidum. Afterlabeling ofep 293on mutagenizedcells witha monoclonalantibodyand tworunsof selection ofunlabeled cells in a FACS IV cell sorter, -5% ofthe clones obtained were defective in theexpressionof theepitope (Table

I).

Mutantclones were easily identified by blotting of the colonies onto

nitrocellulose

filtersandlabelingwith iodinatedantibody (Figure 2). The interestinthese mutants resides inapotential function ofep 293 incell aggregation, ashas beensuggestedbythe block- 2530

ing ofintercellular adhesion by Fab of a monoclonal antibody specificforep 293(Todaetal., 1984a).The structure ofep 293 is highly specific for P. pallidum since even cells of a related organism, D. discoideum, do not react with antibodies against this epitope (Todaetal., 1984a). Thus oneprerequisite for ep 293 to serve in species-specific cell recognition is fulfilled.

Inthe present work we have found a strictcorrelationbetween defects inep 293 expression andaberrationsin cellaggregation under thefollowingconditions. (i) In most of the mutant strains ep 293 expression was delayed rather thancompletelyinhibited;

aggregation of the mutants was atypical in that no or veryshort steams ofcohering cells were formed around the aggregation centers. (ii)Insomesubclonesderived from an unstable mutant, ep 293 wasalmost completely suppressed and,accordingly,only very few rudimentaryaggregates were formed. (iii) By sexual reproduction recombinant progeny strains were obtained which

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Polysphondyliumcarbohydratemutants

rPN60183

-m

^PN600

W :4

200

^-

116

^-

93

-

64

^- ^up-

45-

kDa

0 6 24 6

hours of starvation

Fig.6. Autoradiograph showing suppression ofep293 in the strongly aggregation-deficient mutant PN60183. Mutant cellswere washedatthelate growth phase, and either used immediately (Oh)orafter 6or24 h of shaking innon-nutrient buffer. For comparison, wild-type PN600 cellswere

harvested after 6 h of starvation. Total cell proteinswere labeled with [1251]mAb293afterSDS-polyacrylamidegel electrophoresis and blotting ontonitrocellulose. 30itgof proteinwereappliedtoeach lane.

exhibited either wild-type or mutant aggregation behavior and showed, respectively, normal ^or defective ep293 expression.

The correlation between defective expression ofep 293 and atypical cell aggregation might be duetoarequirement for this carbohydrate structure in normal aggregation. However, it is possiblethattherelationshipisindirect,i.e., aggregation behavior andep293expression mightbecontrolledbycommonregulator

genes thatareinvolved in theoverallprogramming of develop-

ment.Inaprevious studywehave reported that wild-type cells containep293 already during thegrowth phase, which suggested thatitwasconstitutive(Todaetal., 1984a). Ifthiswerethecase,

regulatory genes would probably not be implicated in ep 293 expression. Themutations wouldmorelikelyaffectanenzyme

directly involved in thesynthesis orprocessingof thecarbohy- drates carrying ep 293. However, the finding that ep 293 is expressedinthemutantsonlyaftermanyhoursofstarvationsug-

gests thataregulatory mechanismexists, andthat thedelayof expression in themutants iscaused by inappropriate timingof the regulation.

Anotherpoint that remains tobe clarified iswhetherexpres-

sionofthewholesetofglycoproteins thatcarryep293 isdelayed inthemutants,orwhether theseproteins pre-existinaform that is devoid ofep293. Since L-fucose, ^a sugar which is typical forcomplex-type carbohydrate chains, occupiesaterminalpos-

ition ofep293(Todaletal., 1984b), processing similartocon-

version of carbohydrates from the high-mannosetothecomplex typemight produce the structure recognized by mAb 293.

Materials and methods

Preparation of themonoclonal antibody and labeling of glycoproteins Monoclonal antibody 25-293-2, abbreviated mAb 293, was prepared as described previously (Toda et al., 1984a, 1984b), and iodinated using the chloramine-T method. SDS-polyacrylamide electrophoresis in 10% gels was carried out according to Laemnili (1970), and transfer to BA 85 nitrocellulosefilters (Schleicher andSchuell, Dassel, FRG) and immunolabeling according to Towbin etal. (1979) as specified by Stadler et al. (1984). For cell-surface labeling, 1 x 107washed cells were suspended in100 ul solution containing 100ygmAb 293 in phosphate- buffered saline (PBS) pH 7.0, and incubated for 15 min on ice. The cells were washed three times with PBS at4°C, incubated for 15minon ice with a 1:50 dilution of FITC-conjugated sheep anti-mouse IgG (Institute Pasteur Production), washedagain and analysed using aFACS IVCell Sorter (Becton-Dickinson).

Suspension cultures

Wild-type strain PN600of P. pallidum and mutants derived from it were grown under continuous illuminationat23 ± 1°C in suspensions of 1 x 1010E.coliB/r cells per ml in 17 mM Soerensen phosphate buffer pH 6.0 in Erlenmeyer flasks on a rotary shaker at 150 r.p.m. (Bozzaro etal., 1978). For starvation at the late growth phase, cells were grown up to densities of 7-8 x 106/ml, at which densities1-2 x 109bacteria/ml were left, washed and resuspended in the phosphate buffer at 1 x 107cells/mil, andshaken as described. The suspension cultures were used for mutagenesis, for the determination of generation times by count- ing the cellsin a hemocytometer, for immunofluorescence labeling and for examin- ing ep 293 expression by SDS-polyacrylamide gel electrophoresis and immunoblotting.

Agar plate cultures

For evaluating the morphology of aggregates and fruiting bodies, wild-type and mutants were cultivated under continuous illumination at 23+1 °C on streaks of E. coli B/r on LP agarcontaining0.1 % lactose, 0.1 % peptone, 2% agar and in someexperiments, as that shown in Figure 3, 17 mM Soerensen phosphate buffer pH 6.0.

Selection of mutants

For mutagenesis by u.v. irradiation or treatment with N-methyl-N-nitro-N- nitrosoguanidine (MNNG), PN600 cells were grown in suspension cultures and washed. Cells were treated with 3 mg/mi MNNG in 17 mM phosphate buffer pH 7.0 at23°C for 15-20min in darkness. This resulted in a relatively high survival of -20%, at which double mutations are expected to be infrequent.

After mutagenesis the cells were washed twice in phosphate buffer containing 0.1 MKCl.Thisrelatively high molarity shrank the cells which appeared enlarged and rounded after MNNG treatment, and greatly increased viability. The washed cells were grown in suspension culturesfor 2 days before selection was started by whichtime they had passed through 2-4 divisions.

Forselection of mutants with defective ep 293 expression, mutagenized cells were labeled with mAb 293 andFITC-conjugated sheep anti-mouse IgG antibodies asdescribed above.Unlabeled cells were sorted out and cloned using a FACS- IV cell sorter equipped with asingle-cell deposition system. 12 x 8 cm microtiter plates without wells were filled with LP agar, the agar was covered by a lawn of E. coli B/r, and 96 droplets containing sorted cells were deposited per plate.

After growth of the cloned cells the colonies formed were blotted onto anitro- cellulose filter, labeled with[125I]mAb293and autoradiographed. Sufficient cells remained on the agar surface after blotting to start new cultures from clones which proved to be defective in antibody binding.

Genetic recombination

Crosses wereperformed using the macrocyst sexual cycle as described by Francis (1980). The mutants made in strain PN600 were crossed with wild-type strain PN100(Francis and Rupar, 1983), and haploid progeny were obtained by pick- ing sori fromindividualgerminating macrocysts.

Acknowledgements

D.Francis received a grant as a guest professor of theMax-Planck-Gesellschaft during the first part of this work. K.Toda was a post-doctoral fellow of the Max- Planck-Gesellschaft.

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Received on I July 1985

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Mutants of Polysphondylium pallidum altered in cell aggregation and in the expression of