Proc. Natl. Acad. Sci. USAVol. 87, pp. 8247-8251, November 1990Genetics

Electrophoretic karyotype for Dictyostelium discoideum(clamped homogeneous electric field electrophoresis/pulse-field gels/slime mold genetics)

EDWARD C. COX*, CATHY D. VOCKE, SONJA WALTER, KEQIN Y. GREGG, AND ERIC S. BAINtDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544-1003

Communicated by J. T. Bonner, August 1, 1990

ABSTRACT This paper reports on the separation of theDictyostelium discoideum chromosomes by pulse-field electro-phoresis and the correlation of the electrophoretic pattern withlinkage groups established by classical genetic methods. In twocommonly used laboratory strains, five chromosome-sizedDNA molecules have been identified. Although the majority ofthe molecular probes used in this study can be unambiguouslyassigned to established linkage groups, the electrophoretickaryotype differs between the closely related strains AX3k andNC4, suggesting that chromosomal fragmentation may haveoccurred during their maintenance and growth. The largestchromosome identified in this study is approximately 9 millionbase pairs. To achieve resolution with molecules of this size,programmed voltage gradients were used in addition to pro-grammed pulse times.

The cellular slime mold Dictyostelium discoideum is widelyused in the study of morphogenesis, cell-cell interaction, andsignal transduction (for recent reviews, see refs. 1 and 2).Extensive genetic studies have helped to define the genesunderlying these processes, and many genes have beenmapped to well-studied linkage groups (reviewed in ref. 3). Aconsiderable number have also been ordered by mitoticrecombination (4, 5). Laboratory strains ofD. discoideum arethought to have seven chromosomes (6, 7), although only sixlinkage groups have been identified (3).To aid in the correlation of linkage group with physical

structure and to develop a rapid and accurate method forstudying genetic fine structure in D. discoideum, we haveexamined the conditions necessary for separation of D.discoideum chromosomal-sized DNA molecules by pulse-field gel electrophoresis (8, 9). We report here on the numberofelectrophoretic species detected by clamped homogeneouselectric field (CHEF) electrophoresis (10) and show, using avariety of molecular probes, that it is possible to correlate thegenetic and physical map, thus enlarging the genetic possi-bilities in this organism.

MATERIALS AND METHODSStrains. D. discoideum AX3k and NC4 were received from

R. Firtel (University of California, San Diego, CA) and J. T.Bonner (Princeton University, Princeton, NJ), respectively.Growth conditions. In most experiments, cells were prop-

agated on 15-cm GYP plates (11) with Escherichia coli B/r asthe food source. Strain AX3k was grown on E. coli B/r oraxenically in HL5 medium (12). Incubation was at 21°C in ahumidified growth chamber or at 21°C in a water bath on arotary shaker. Cells were starved by plating well-washedcells at a density of approximately S x 104 amoebae per mm2on 2% agar plates (20 g of agar per liter of distilled deionizedwater).

Cell Preparation. Cells were harvested at various stages ofgrowth and development, washed several times at roomtemperature in phosphate buffer (13), and mixed at 390C withan equal volume of FMC InCert agarose. For most of theexperiments reported here, cells were allowed to develop tothe loose-mound stage before they were harvested. As wewill discuss in Results, DNA prepared otherwise gives verypoor resolution. The final cell concentration varied between5 x 108 and 2 x 109 cells per ml. Aliquots (25 s.l) were pipettedinto rectangular molds spaced to coincide with the wells offlat-bottom 96-well plates (Coming 25860). After cooling, themold contents ("plugs") were pushed into the wells, whichcontained 100 ,ud of 0.5 M EDTA (pH 9.5). After incubationat room temperature for 1 hr, 100 tul of 0.5 M EDTA (pH 9.5)containing 2% sodium lauryl sarcosinate and 2 mg of pro-teinase K (BRL) per ml was added. The plates were sealedand incubated at 50°C for approximately 24 hr. The plugswere stored at 4°C. Plugs prepared in this way give repro-ducible electrophoretic patterns for more than 1 year.

Electrophoresis. For running gels, 0.8% "electrophoresisgrade" agarose (BRL) was dissolved in running buffer (27mM Tris base/27 mM boric acid/0.75 mM EDTA, pH 8.5).Agarose plugs were sealed in the wells ofthe running gel with0.8% FMC InCert agarose dissolved in running buffer. Elec-trophoresis was for 240 hr at 10°C in running buffer with aCHEF apparatus of the dimensions described by Chu,Volrath, and Davis (10). The starting voltage and amperagewere 1.85 V/cm and 50 mA. The pulse time was increasedlinearly from 2000 to 9600 sec during the run, and the voltagewas decreased from 1.85 V/cm to 1.48 V/cm at a pulse timeof 7000 sec.Molecular Probes. With the exception of mitochondrial

DNA (mtDNA), rRNA-encoding DNA (rDNA), and thephage Agt11 inserts, protein-coding sequences were cut andpurified from vectors carrying cDNA clones (Table 1). ThetRNAval probe was the one unique 250-base-pair (bp)HindIII-BamHI subfragment described in ref. 14. The ribo-somal probe was homologous to the 5.8S, 17S, and 26S RNA(fragment VII of ref. 35). The mitochondrial probe was theentire plasmid containing a mtDNA insert. Probes C1 to C17were isolated from a Agtll cDNA library prepared from cellsstarved for 8 hr. Inserts were amplified from single Agtllplaques by the polymerase chain reaction with Agtll forwardand reverse primers (New England Biolabs).Southern Blots. Agarose gels were stained with ethidium

bromide and photographed. The DNA was depurinated inacid, nicked in alkali, and transferred to nylon membranes asdescribed by Church and Gilbert (36). After the DNA wascross-linked to the nylon by exposure to a high-intensity

Abbreviations: CHEF, clamped homogeneous electric field; rDNA,rRNA-encoding DNA; tRNAVal, tRNAvaIGUUB3s3; Gf,8 guanine nu-cleotide-binding regulatory protein ,32 subunit; CARl, cyclic AMPreceptor 1; CP1 and CP2, cysteine proteinases 1 and 2; PDE,phosphodiesterase; PsA, glycoprotein PsA.*To whom reprint requests should be addressed.tPresent address: University of California San Francisco School ofMedicine, San Francisco, CA 94143-0724.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 87 (1990)

Table 1. DNA probes used in this study

Probe Linkage group

Name Abbreviation Ref. No. Ref.

a-Actinin 15 I 16Glycoprotein PsA PsA 17 I 18Actin 15 19 I, II 20Discoidin I 21 II 22Cyclic AMP receptor 1 CAR1 23 II A*G protein ,82 subunit GP3 24 II A*tRNAvaJGUUB353 tRNAval 25 III or VI 14Phosphodiesterase PDE 26 IV A*Myosin heavy chain 27, 28 IV 29Severin 30 VII 31Contact site A 32 VII BtCysteine proteinase I CP1 33 VII A*Cysteine proteinase 2 CP2 34 CtC1.,C17 Ct CtrDNA fragment VII 35 CtmtDNA D§ Ct

G protein, guanine nucleotide-binding regulatory protein.*A, D. Welker, personal communication.tB, E. Wallraff, personal communication.tC, this work.§D, S. Alexander, personal communication.

ultraviolet light, the membranes were blocked and probed(36). 32P-labeled probes were prepared by nick-translation(37). Probing was at 60°C followed by washing at 65°C underthe Church and Gilbert conditions. Membranes were thenexposed to x-ray film at -20°C before development.

RESULTSWe knew from previous experiments with Polysphondyliumpallidum that long pulse times and correspondingly long runtimes would be required to separate chromosome-sized D.discoideum DNA molecules. In both species, we found thatDNA from vegetative cells could be made to enter 0.8% gels,but that it did so as a single diffuse band, somewhat like theresults reported by Cole and Williams (38). The reasons forthis are not known, but by using starved cells, D. discoideumAX3k and NC4 DNA could be resolved into seven majorbands as shown in Fig. 1. With the exceptions noted below,individual probes hybridized to unique bands, to DNA re-

a D. discoideum AX3k

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maining in the sample plugs, and to fragmented chromosomalDNA running near the mtDNA and rDNA bands. NC4 bandnumber 1 was labeled by all probes, although, because of thevarious exposure times used to produce the Southern blotsfor Figs. 1 and 2, this is only apparent for the tRNA, contactsite A, and actin probes. This result suggests that this regionof the NC4 gel contains sequences from the entire genome.The largest band in AX3k does not exhibit this behavior.Finally, the results with AX3k were the same whether or notthe cells were propagated on E. coli, and the electrophoreticpatterns in both strains have proven to be highly reproduciblefrom preparation to preparation over an 18-month period.The majority of the chromosomal-sized DNA molecules

hybridized to our probes unambiguously and can be assignedto known linkage groups (Table 2). There is good agreementbetween the published linkage map and the electrophoretickaryotype outlined here (compare Table 1 with Table 2). Thecorrespondence is especially good for AX3k. In four cases-actin, tRNAv1, CP2, and probe C5-there are copies ofclosely related or identical sequences on one or more addi-tional chromosomes.We found that the GP (see Table 1) and PDE probes

hybridized to NC4 bands 4 and 5, respectively, whereas theyhybridized to bands 1 and 3 in AX3k. Representative dataillustrating this point for GP are shown in Fig. 2, where thecyclic AMP receptor CAR1, GP, and discoidin I probes havebeen hybridized to AX3k and NC4. Although CAR1 anddiscoidin both clearly mapped to band 1 in AX3k (linkagegroup II) and band 6 in NC4 (also linkage group II), GPhybridized to band 4 of NC4.Perhaps most interestingly, known linkage group II mark-

ers hybridized to the largest band in AX3k and to the smallestin NC4. The NC4 result was unexpected since, as we pointout in Discussion, linkage group II appears by genetic criteriato be the largest D. discoideum chromosome.The two fastest AX3k species, bands 6 and 7, were not

always well resolved and coded for rDNA and mtDNAsequences, respectively, along with variable amounts ofdegraded chromosomal DNA (Fig. 1). In NC4, rDNA andmtDNA sequences had approximately the same electropho-retic mobility. The intensities of bands 6 and 7 varied frompreparation to preparation and were independent of whetheror not AX3k was grown axenically. The reasons for this arenot understood.

b D. discoideum NC4

1

2

3

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FIG. 1. Dictyostelium discoideum AX3k (a) and NC4 (b). Agarose gels were stained with ethidium bromide and then transferred to nylonmembranes and probed as described. The sources ofthe probes are listed in Table 1. Schizosaccharomycespombe (Sp) molecular weight markers(39) are shown on the right (lane Sp); sizes are shown in Mbp. The band numbers correspond to those in Table 2 and are shown to the left ofa and b.

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8248 Genetics: Cox et al.

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Proc. Natl. Acad. Sci. USA 87 (1990) 8249

Table 2. Localization of probes to electrophoretic bandsD. discoideum AX3k D. discoideum NC4

Proposed ProposedBand Probe(s) linkage group Band Probe(s) linkage group*

1 Actin, CARL, discoidin 1, CP2, Gp, tRNAvaI II 12 tRNAVal, CP2 III or VI 2 tRNAval, CP2 III or VI3 MHC, contact site A, CP1, severin, CS, C10, IV, VII 3 MHC, contact site A, CP1, severin, IV, VII

achin, PDE CS, C104 a-Actinin, actin, PsA, C4 I 4 a-Actinin, actin, PsA, C4, G,8 I5 C1, C6 III or VI 5 C1, C2, CS, C6, PDE III or VI6 rDNA 6 Actin, CAR1, discoidin 1 II7 mtDNA - 7 rDNA, mtDNA

*See Discussion for those probes (PDE, C5, G/3) in NC4 that are at variance with these assignments. MHC, myosin heavy chain.

We have also mapped 17 randomly chosen cDNA insertsisolated from developing D. discoideum cells. All of themmapped to at least one electrophoretic band (a selection isshown in Table 2). Probes C1 and C6 were the only markersdefining band 5 in AX3k, which is likely therefore to be eitherlinkage group III or VI (see Discussion). This conclusion isconsistent with the NC4 results, where C1 and C6 alsohybridized to band 5.The largest chromosome-sized molecule resolved in this

study is approximately 9 million base pairs (9 megabase pairsor 9 Mbp; Fig. 3) close to the upper limits for pulse-field gelelectrophoresis. Therefore, it is possible that we have notsucceeded in our attempts to electrophorese all of the chro-mosomes into the gel. We do not think this is likely, however,because all of the 32 probes listed in Table 1, including thoseisolated at random from a cDNA library, hybridized to atleast one of the bands. Significantly, with the exception ofmtDNA and rDNA, no probes labeled only the DNA remain-ing in the plug, or in the low molecular weight region,suggesting that we have located the entire genome.The electrophoresis conditions required to achieve the

resolution reported here are very sensitive to such variablesas buffer composition, agarose source, pulse times, andvoltage gradients. Although we have not found it possible toexplore all of these parameters systematically, it is clear thatgood resolution depends on both a linearly increasing pulsetime and a decreasing voltage gradient. For the resultsreported here, a stepped voltage gradient was used; however,a linearly decreasing voltage gradient, ramped between thetwo extremes we have used, was equally effective. However,when the voltage gradient was constant for the duration oftherun, the two largest chromosomes apparently sheared intosmaller fragments. By controlling these parameters carefully,the electrophoretic karyotype was reproducible both from

a AX3k b NC4pI ,W

day to day and from one preparation to another. The fluo-rescent intensity of bands 1-5 (AX3k) and 1-6 (NC4) was alsoreproducible, suggesting that not all bands were present inequimolar amounts. It is also possible that there were sub-stantial differences in ethidium bromide binding betweenbands, given the very high A+T content of D. discoideumDNA.The conditions for successful preparation ofchromosomal-

sized DNA molecules in D. discoideum depend on the use ofstarved cells and high EDTA concentrations prior to theaddition of detergent. Starvation may be important becausethe DNA of vegetative cells can be expected to contain manyreplicating forks per chromosome. Such a structure approx-imates circular DNA, which behaves anomalously in pulse-field gels (40). When detergent and EDTA were addedtogether during sample preparation (41), D. discoideum DNAwas degraded, perhaps because lysozomal nucleases arereleased by detergent before Mg2` ions are effectively che-lated.

DISCUSSIONThe genetic literature suggests that there are six linkagegroups in commonly used D. discoideum strains (3), althoughcytological examination of metaphase preparations revealsseven (6, 7). Our results clearly show six, although it isdifficult to rule out that there is a seventh, even with the useof randomly chosen cDNA probes, since there could be aseventh transcriptionally silent chromosome. It seems likelyto us, given the data presented here for AX3k and NC4, thatdiscrepancies in the literature between the linkage map andkaryotype could be due to strain differences. This view isreinforced by our recent finding that widely used strains fromother laboratories, particularly axenic derivatives of NC4,have surprisingly different electrophoretic patterns (unpub-lished data). Thus, it seems possible that there is strong

a b

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FIG. 2. Mapping the GP3 protein, a cyclic AMP receptor, anddiscoidin I to the AX3k and NC4 chromosomes. Band numbers referto Fig. 1 and Table 2 and are shown to the left of each gel.

, 3.6

10 20 30 40 10 20 30 40

Distance (mm)FIG. 3. Apparent molecular masses of D. discoideum AX3k (a)

(sum = 34 Mbp) and NC4 (b) (sum = 27.6 Mbp) chromosomal DNA.The apparent molecular mass in Mbp is plotted as a function of thedistance migrated. *, S. pombe markers.

Genetics: Cox et al.

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Proc. Natl. Acad. Sci. USA 87 (1990)

selection in the laboratory for rearrangements, chromosomalfusions, and gene duplications.With the exceptions noted below, our results agree well

with the published linkage map (Tables 1 and 2). However,we find that CP2 and our tRNAval probe hybridize to band 1(linkage group II) in AX3k as well as to the expected band 2(linkage group III or VI). CP2 was isolated by hybridizationat low stringency from a cDNA library by using a CP1 probe(34) and is a member of a larger family of cysteine proteinases(42, 43). Thus CP2 could either be duplicated in AX3k as wehave implied in Table 2, or we may be detecting othermembers of this family. The latter interpretation seems,however, less likely because of the high-stringency hybrid-ization conditions used in our experiments.Dingermann et al. (44) probed HindIll digests of a variety

ofD. discoideum strains with the unique tRNAv' probe usedin this study, finding that it hybridized to a single band in eachcase. These results are consistent with those we have ob-tained with NC4. They are also consistent with our AX3kresults, provided that the apparent duplication we havedocumented here contains the HindIII sites used in theirstudy.Of the more than 100 genes located on six linkage groups

(3), 31 are on linkage group II. This is the largest chromo-some-sized molecule identified in AX3k and is the expectedresult if map size is related to DNA content. In NC4,however, most linkage group II markers map to band 6. Thesimplest explanation for this is the following. Our NC4 strainis from J. T. Bonner's laboratory, where it has been propa-gated by mass culture for nearly 50 years. It seems likely thatduring this time part of linkage group II has fragmented andnow carries approximately one-third of the chromosome,while the remainder migrates elsewhere. As we probe NC4with additional markers from linkage group II, we mightexpect to find the remainder of this chromosome, perhapscomigrating with band 2 (since tRNAvaI hybridizes to linkagegroup IT in AX3k) or band 4 (the location of G,3 in NC4). Asimilar explanation may explain probe C5 and phosphodi-esterase. Note that although they hybridize to different bandsin the two strains, they appear to be linked, again suggestingconsiderable chromosomal rearrangement.The actin-coding sequence used in our experiments is a

member of a family of approximately 20 genes, each of whichis highly conserved at the nucleotide level (45). It hybridizesto at least three chromosomes in AX3k and two in NC4. Therestriction fragment length polymorphism studies of Welkeret al. (20) localized actin genes to linkage groups I and II.These workers utilized unique actin 5' and 3' noncodingsequences in their experiments. It is not surprising, then, thatwe have identified sequences consistent with their data aswell as additional actin homologies on linkage groups IV andVII. Clearly, however, members of this large family are notdispersed over the entire genome in either strain.The D. discoideum nuclear DNA complexity has been

reported to be 11-12 times the complexity of Escherichia coli(45), which is now known to be 4.7 Mbp (46). Thus, the D.discoideum genome size is 52-56 Mbp. Seventeen percent ofthe nuclear DNA is rDNA (47,48), and there is good evidencethat it is extrachromosomal. The total non-rDNA genome istherefore 44-46 Mbp. Although Fig. 2 is clearly an extrap-olation for which we do not have adequate standards ortheory to guide us, by assuming that the mass of eachchromosomal band falls roughly on the exponential curves inFig. 2, and by assuming that band 3 in both strains is anunresolved pair of chromosomes (linkage groups IV and VII),we arrive at estimates of 40.7 Mbp for AX3k and 33.7 Mbpfor NC4. Given the uncertainties in the original measure-ments of genome size, and the limitations of Fig. 2, thisagreement with earlier values is of the right order. The truegenome size of both strains will become evident as we

complete large-scale restriction maps ofeach electrophoreticband.The above arguments, which lead us to believe we have

resolved all of the D. discoideum genome, are further sup-ported by the observation that all of our probes hybridize toone or more bands. We have used 32 probes isolated fromcDNA libraries. Of these, 15 have been sequenced andcorrespond to well-studied genes or gene products, while 17are of unknown function, but nonetheless represent tran-scribed sequences. Of these 32 probes, 28 hybridize to oneband, whereas 4 hybridize to two or more. Significantly, nonehybridizes only to the DNA that has not entered the gel orruns in the low molecular weight region. If an additionalchromosome exists, it must code for very few transcripts.

We are indebted to the many research groups who supplied themolecular probes used in these experiments. We thank S. Cohen andW. F. Loomis for the Agtll library. Dennis Welker was particularlygenerous with his time and advice. This research was supported byNational Institutes of Health Grant HD23301 and National ScienceFoundation Grant DCB-8616302 to E.C.C., by a National Institutesof Health National Research Service Award Grant GM11730 toC.D.V., and by a Genetics Society of America Summer Fellowshipto E.B.

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