Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418

PLASMID

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Selection of Dictyostelium Vector Maintenance

discoideum Transformants and Analysis of Using Live Bacteria Resistant to G418

JOANNE E. HUGHES, GREGORY J. PODGORSKI, AND DENNIS L. WELKER’ Molecular

Biology Program, Biology Department,

Utah State University,

Logan, Utah 84322-5500

Received October 2 1, 199 I ; revised January 20, 1992 A protocol that allows the rapid isolation and growth of large numbers of independent G4 1Sresistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies, Discovery that aggregation of nontransformants was inhibited at a nonselective level of G4 18 (25 to 35 *g/ml) led to the development ofa vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddpl and Ddp2. We conclude that the origin of replication of plasmid Ddpl does not alone confer stable maintenance and thus, Ddp 1 must bear additional sequences required for its own maintcnancc. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3’ end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified. 0 1992 Acadcmx Press. Inc.

Dictyostelium discoideum is a eukaryotic microorganism with a life cycle that alternates between single-celled and multicellular stages. Following nutrient deprivation, the amoebae aggregate and undergo a program of differentiation and development. D. discoideum has been extensively utilized to explore mechanisms of gene expression and the role of signal transduction systems in development (Dottin et al., 1991), morphogenesis (Ceccarelli et al., 199 I), cell motility (Devreotes and Zigmond, 1988), and the structure and function of eukaryotic extrachromosomal DNA elements (Farrer et al., 1988; Orii et al., 1989; Jensen et al., 1989; Leiting et al., 1990; Slade et al., 1990; Gurniak et al., 1990; Yin and Welker, 1992). Transformation of D. discoidcum has relied heavily on the use of axenically grown amoe’ To whom correspondence should be addressed. 0147-619X/92

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Copyright 0 1992 by Academx Press, Inc. All rights of rcproducl~on on any form reserved.

bae for the selection of transformed cells and their subsequent growth (Nellen et al., 1984; Howard et al., 1988; Egelhoff et al., 1989; Chang et al., 1989; Kalpaxis et al., 1990; Knecht et al., 1990). Isolation of independent transformants is time consuming using axenically growing cells and, because D. discoideum development typically occurs at an air-substrate interface, growth of transformants in liquid axenic medium does not allow direct screening for developmental effects. Analysis of the developmental phenotype of large numbers of independently derived transformants will be particularly important in experiments designed to identify cloned DN.4s that complement developmental mutations. To overcome these limitations, we developed a procedure for the isolation and continuous growth of G4 18-resistant transformants under selective pressure on the surface of agar media. Our group has investigated Dictyostelium plasmid DNAs. The genus harbors the most

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complex group of nuclear eukaryotic plasmids described (Metz et al., 1983; Noegel et al., 1985; Hughes et al., 1988; Orii et al., 1989; Leiting et al., 1990; Slade et al., 1990; Gurniak et al., 1990; Yin and Welker, 1992). During the development of the new selection procedure we found that the aggregation of nontransformed cells was blocked on agar containing nonselective concentrations of G4 18, while that of G4 18-resistant transformants was unaffected. This observation led us to develop a sensitive assay for vector maintenance that allows the direct visual screening of colonies to identify those that have lost vector DNA. The assaycan be used to identify plasmid genes and sequences involved in vector maintenance. We report findings obtained with the vector maintenance assayconcerning the stability of extrachromosomal and integrating vectors derived from the Ddp 1 and Ddp2 native D. discoideum plasmids.

DNA constructs were E. coli CES20 1 and II. coli SURE (Stratagene). DNA Isolation and Churucteriaation D. discoideum total nuclear DNA and plasmid DNAs were isolated, and the presence of plasmid DNA in individual colonies of D. discoideum cells was determined as previously described (Welker et al., 1985; Hughes et al., 1988; Hughes and Welker, 1988). The copy number of plasmid p7d2 was determined using a densitometer to compare the plasmid band in restriction enzymedigested nuclear DNA sampleswith neighboring bands from the ribosomal DNA (Hughes and Welker, 1989). The copy numbers of other plasmids were estimated by comparison to p7d2 or to rDNA bands in restriction enzyme-digested total nuclear DNA of AX4 transformants. Plasmid copy numbers may be higher in HUD205 transformants. Electrophoresis, Blotting, and Detection

MATERIALS

AND METHODS

Restriction enzyme-digested total nuclear DNA and purified plasmid DNA were anaStrains and Culture Conditions lyzed using conventional electrophoresis on Nonaxenic D. discoideum cultures were 0.8% agarose gels. Undigested total nuclear grown on DM agar (Podgorski and Deering, DNA and plasmid DNA were also analyzed 1980) with or without the addition of the anti- using the Bio-Rad CHEF DRII pulsed-field biotic G4 18 (added after the medium was au- electrophoresis system. Bio-Rad DNA grade toclaved). On medium lacking G4 18 the food agarose gels at a concentration of 1% were source was Escherichia coli B/r and on G4 18- run at 16OVwith a switch time of 45 s for 20 containing medium, it was the E. coli B/r h at 14°C. The samples for the pulsed-field transformant ECU1 that carries plasmid gels were purified on CsCl gradients and pUC4K (Pharmacia) conferring resistance to loaded into the wells as aliquots with loading G4 18. We also used Klebsiella aerogenesas a buffer. Commercially obtained agaroseplugs recipient for plasmid pUC4K and grew these containing concatemers of X DNA (Cloncells on SM agar (Sussman, 1966). Axenic D. tech) were used as size standards. Agarose discoideum cultures were grown in HL5 me- gels were blotted onto either nitrocellulose dium (Nellen et al., 1984) with or without membranes (MSI) using the Southern blotG418 (added as a sterile solution in 10 mM ting procedure (Southern, 1975), or onto Hepes, pH 7.2). All D. discoideum strains charged nylon membranes (MSI) using the were grown at 2 1 + 1“C. Plasmid-free D. dis- alkaline blotting procedure (Reed and Mann, coideum recipients used in transformations 1985). Probe DNAs were labeled either were AX4 (genotype axeAl, axeB1) and with [32P]dATP or with digoxigenin-dUTP HUD205 (genotypes axeAl, axeB1, tsgA1, (Boehringer-Mannheim). Hybridization of bwnA1, and ebrA1) (Jensen et al., 1989). Bac- probe DNAs to blots was carried out in hyterial recipient strains used to prepare vector bridization buffer at 68°C in the absence of

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formamide or at 42°C in 50% formamide. Digoxigenin-labeled probe DNAs were detected using the Lumi-Phos 530 chemiluminescent detection procedure (BoehringerMannheim). Blots were exposed to Kodak XOMat AR X-ray film, with an intensifying screen in the case of 32P-labeledprobes.

AND

WELKER

A. Noegel. The plasmid pDA27a was modified by inserting an EcoRI fragment carrying a gene encoding resistance to G418 into the EcoRI site between the Ddp2 sequencesand the bacterial vector pUC 19. The resultant plasmid was designated p 103d2.

Transformation and Selection Plasmids

D. discoideum transformations were carried out using either the calcium phosphate The Ddp2-derived plasmids constructed in precipitation technique (Nellen et al., 1984) our laboratory and used in this study are or electroporation (Howard et al., 1988; shown in Fig. 1. The recombinant plasmid Dynes and Firtel, 1989). After transformap7d2 (Hughes et al., 1988; Hughes and tion the D. discoideum cells were incubated Welker, 1989) consists of the entire Ddp2 seovernight in liquid HL5, followed by a secquence with a gene encoding resistance to ond overnight incubation in HL5 with 15 pg G4 18 inserted in the unique San site. PlasG418/ml. The cells were then harvested and mid p27d2 (Hughes et al., 1988) differs from inoculated to plates containing 80 pg G4 18/ p7d2 in that a 1.I-kb Hind111 fragment, inml along with E. coli strain ECU 1. When decluding one element of the inverted repeat, termining G418 concentrations for use in has been deleted. The shuttle vector p3 1d2 agar media it must be kept in mind that the consists of the entire Ddp2 sequence in the toxicity of the G4 18 can vary from one lot to form of p7d2, with pGEM3Z inserted as a another and, in addition, the toxicity can be San-BamHI fragment at the 3’ end of the affected by changes in media components, G418 resistance gene cassette. The shuttle for example, by different lots of agar. There is vectors p87d2 and p7 1d2 consist of Ddp2 in also strain specific variation in native resiswhich the 1.2-kb HindIII-S&I region has tance to G4 18. been deleted and replaced by HindIII-Sandigested p60 (seebelow). The only difference VectorMaintenance between these two plasmids is the deletion of a small fragment containing the EcoRI site Vector maintenance assays were carried between the G418 resistance gene cassette out using, as the initial inoculum, cells grown and pGEM3Z in p87d2. The shuttle vector on plates containing 80 pg G4 1g/ml. These p30d2 consists of p27d2 into which cells were harvested and inoculated at 3 X 1O4 pGEM3Z has been inserted as a Hind111frag- cells/plate onto DM medium without G418 ment. The shuttle vector p86d2 was con- (mass plates) and at 50 cells/plate onto DM structed from p3 ld2 by deleting the 1.2-kb medium and onto DM medium containing HindIII-San region of Ddp2. The integrating 25-35 pg G418/ml (assay plates). Every 3 vector p60 was constructed by inserting the days (approximately 20 cell generations) the San-EcoRI fragment containing a gene con- nonselectively grown cells from a mass plate ferring resistance to G418 (Nellen et al., were used to set up new mass plates and new 1984) into the bacterial vector pGEM3Z. assayplates. Colonies appearing on the assay The plasmid p88dl consists of p60 into plates with and without G418 were counted which a 2.0-kb Hind111 fragment, identified daily from Day 3 to Day 7 after inoculation as containing the Ddpl origin of replication and the number of these colonies capable of (Ahern et al., 1988), has been inserted. The forming fruiting bodies on the plates with Ddp2-derivatives pnDEA1, pnDEI1, pnD- G418 and without G418 was scored on the dp2, pnDHI1, pnDAX4, and pDA27a (Leit- seventh day. The ratio of the latter numbers ing et al., 1990) were kindly provided by Dr. is plotted as the y-axis in Figs. 2, 3, and 4.

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RESULTS

Selection qf D. discoideum Tran@rmants To select G4 1S-resistant Dictyosleliurn transformants on agar media we begin with axenically grown plasmid-free recipient cells, introduce transforming DNA by electroporation or calcium phosphate precipitation, allow an overnight expression time in axenic medium, expose the cells overnight to 15 pg G4 18/ml in axenic medium, recover the cells from the axenic medium, and then inoculate them onto DM agar containing 80 pg G4 18/ ml in association with the E. coli B/r pUC4K transformant ECU 1. Under these conditions transformant colonies appear 3 to 5 days after inoculation and grow and develop well. A few slow growing colonies occasionally appear, which we infer are nontransformed cells. These typically do not grow larger than 1 mm in diameter and do not form fruiting bodies. Using this method transformation frequencies of 1O-4to 10e6are routinely obtained, although this varies due to recipient strain- and vector-dependent effects. The ability to select transformants on agar medium facilitates quantitative characterizations of the efficiency of transformation of different recipient strains. In particular, we uncovered a difference in the frequencies with which recipient strains AX4 and HUD205 were transformed by integrating vectors. AX4 and HUD205 transformed equally well with autonomously replicating vectors. In typical experiments with autonomously replicating vectors, frequencies between 1Om5and 1OP6transformants per input recipient cell were obtained and in some experiments frequencies of lop3 were obtained. With integrating vectors AX4 also typically had a transformation frequency in the 1Oe5to 10e6range. However HUD205 had a transformation frequency in the 1Om6 to lo-’ range with integrating vectors and we failed to obtain any transformants in some experiments. Since HUD205 was similar to AX4 in its ability to be transformed with autonomously replicating vectors, we attribute this effect to the decreased ability of HUD205 to integrate

nonautonomously replicating vectors into its chromosomes and not to a difference in the ability of HUD205 to take up vector DNA or to survive or divide prior to inoculation of the transformed cell population on agar plates. The Vector Maintenance Assay While analyzing the G418 sensitivity of nontransformed recipient strains, we observed that development of fruiting bodies is inhibited at G4 18 concentrations that have little effect on vegetative growth. Aggregation is blocked, leaving the colonies either with a flat aggregatelessphenotype or with tipless aggregates. In contrast, transformant colonies form fruiting bodies at both low and selective levels of G418. In particular, a transformant containing a single integrated copy of the G4 18 gene developed at up to 120 pg G4 18/ml. Thus the distinction between colonies that carry a G4 18 resistance vector and those that do not is easily made by a simple visual test of whether or not development proceeds past aggregation. This allows us to follow the maintenance or loss of vectors during nonselective growth by analyzing the development of fruiting bodies in colonies formed on plates containing a low concentration of G418 (25 pg/ml for AX4 transformants, 35 pg/ml for HUD205 transformants). Plasmid Maintenance Slows Cell Growth To determine whether the presence of a vector decreasesthe growth rate of a transformant relative to that of a plasmid-free cell, we mixed p7d2 transformant HUD722 with its parental recipient strain HUD205 at a two to one ratio of HUD722 to HUD205 cells. After 100 generations of nonselective growth, only 3 1% of colonies were capable of developing on plates with a low G4 18 concentration. On plates without G418, 99% of the colonies formed fruiting bodies. In a concurrent experiment using the sameinitial HUD722 population, only 4 nondeveloping colonies were seen on plates containing a low G418 con-

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R

FIG. 1. Maps of Ddp2 and vectors derived from Ddp2 in this work; other Ddp2-derived vectors (pnDEA1, pnDEI1, pnDdp2, pnDHI1, pnDAX4, and pDA27a) were gifts from A. Noegel (Leiting et al., 1990). The Ddp2 inverted repeat elements are indicated by the closed arrows and the Ddp2 open reading frame by the open arrow. The light dashed line indicates pGEM3Z or pUC 19 (p103d2) sequencesand the heavy dashed line the G418 resistance gene cassette.The small arrow shows the direction of transcription of the G4 18 resistance gene. Restriction enzyme sites indicated are EcoRI (R), Hind111(H), and Safl (S).

centration out of a total of 2504 (0.16%) assayed over a period of 340 generations. Clearly, p7d2 is almost completely stable in HUD722 and the decreasedproportion ofcolonies capable of developing in the passaged mixed population reflects a growth disadvantage due to the presence of p7d2. We infer that native plasmids also decrease cell growth. Thus in the experiments reported below a portion of the increase in the number of colonies that cannot form fruiting bodies on plates containing a low concentration of

G4 18 reflects the growth advantage of plasmid-free cells.

Maintenance of Ddp2 Extrachromosomal Vectors The native plasmid Ddp2 is 5.8 kb in length, present at approximately 250 copies per haploid nucleus, and extremely stable (Chang et al., 1990; Leiting et al., 1990; Hughes and Welker, 1989). The plasmid sequence reveals an inverted repeat made up of

TRANSFORMATION

AND VECTOR MAINTENANCE

G 1

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FIG. 2. Maintenance of DdpZ-based extrachromosomal vectors: (A) p7d2 in HUD722 (0) (HUD205 transformant); (B) p27d2 in HUD725 (0) and HUD748 (0) (HUD205 transformants) and in HUD747 (+) (AX4 transformant); (C) p31d2 in HUD750 (0) (HUD205 transformant) and in HUD749 (+) (AX4 transformant); (D) p30d2 in HUD757 (0) and HUD947 (0) (HUD205 transformants) and in HUD755 (+), HUD766 (A), and HUD948 @) (AX4 transformants); (E) p86d2 in HUD775 (0) and HUD776 (0) (HUD205 transformants) and in HUD767 (+) and HUD774 (A) (AX4 transformants); (F) pnDEA1 in HUD935 (0) HUD943 (O), and HUD944 (A) (HUD205 transformants) and in HUD934 (+), HUD945 (x), and HUD946 (m) (AX4 transformants); (G) p71d2 in HUD726 (0) and HUD752 (0) (HUD205 transformants) and in HUD727 (+) and HUD75 1 (A) (AX4 transformants); and (H) p87d2 in HUD769 (0) and HUD778 (0) (HUD205 transformants) and in HUD754 (+), HUD768 (A), and HUD777 (w) (AX4 transformants). The x-axis indicates the number of generations the cells were grown in the absenceof G418, and the y-axis indicates the ratio of the number of colonies able to form fruiting bodies in the population on medium containing G4 18 divided by the number of colonies able to form fruiting bodies in the population on medium lacking G4 18.

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HUGHES, PODGORSKI, AND WELKER

D 1

1

+

lo

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FIG. 3. Maintenance ofintegrating vectors: (A) pnDdp2 in HUD905 (0) (HUD205 transformant) and in HUD900 (+) and HUD909 (A) (AX4 transformants); (B) pnDHI1 in HUD907 (0) (HUD205 transfor-

TRANSFORMATION

AND VECTOR MAINTENANCE

two 0.5kb elements and a 2.7-kb open reading frame (ORF) encoding the rep gene. The construction of vectors from native plasmids inevitably involves disruption of plasmid structure. We used the maintenance assayto determine the effect of such disruptions on the vector’s ability to maintain itself. As described above, vector p7d2 was almost never lost. This vector carries all of the Ddp2 sequence, including the entire inverted repeat, with the G418 resistance cassette inserted at the unique sun site (Fig. 1). Two vectors derived from p7d2 were slightly less stable. Deletion of one element of the inverted repeat produced vector p27d2 (Fig. 1). Over the course of 400 generations of nonselective growth of the p27d2 transformant HUD725, 118 of 2670 colonies (4.4%) examined on plates with a low G418 concentration were blocked in early aggregation. The shuttle vector p31d2 differs from p7d2 by having the bacterial vector pGEM3Z inserted adjacent to the G4 18 resistance gene cassette(Fig. 1) without altering any other features of the plasmid. The p3 ld2 transformant HUD750 yielded 22 AGG- colonies out of 2 174 (1%) analyzed over the course of 240 generations. In both cases,equivalent numbers ofcolonies grown on medium lacking G4 18 were examined and none were developmentally defective. Thus all three of these constructs produced only minor disruption of the plasmid’s ability to maintain itself. No differences were observed between transformants derived from strain AX4 and from strain HUD205. Insertion of pGEM3Z and concurrent deletion of one element of the Ddp2 inverted repeat generated shuttle vectors with markedly decreased stability compared to p7d2, p27d2, and p31d2. The independently constructed vectors p30d2, p71d2, p86d2, and

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p87d2 (Fig. I) all showed an easily discernible decreasein the proportion of AGGf colonies as the transformant populations were grown for longer times on nonselective medium (Fig. 2). This was true whether the transformant was derived from HUD205 or from AX4, but with those derived from HUD205 the loss was more rapid. In HUD205 transformants carrying these vectors approximately 50% of the cells had lost the plasmid after 160 generations of nonselective growth. Since these vectors were independently constructed, the observed instability cannot reflect unanticipated mutations in the Ddp2 component of the vectors but must reflect the combined effect of the loss of one element of the repeat and the presence of the bacterial plasmid DNA. A comparison of the maintenance of p30d2 and p86d2 with that of p7 ld2 and p87d2 illustrates that the orientation of the highly transcribed G418 resistance gene relative to that of the Ddp2 rep ORF is not an important factor. The vector pnDEA1 (Leiting et al., 1990) was obtained from Dr. A. Noegel and also examined using this maintenance assay.Like p30d2, p71d2, p86d2, and p87d2, this plasmid carries only one element of the Ddp2 inverted repeat, a G418 resistance gene cassette and a bacterial vector (pUCl9). Like these four vectors, pnDEA1 had an easily discernible decrease of AGG+ colonies after nonselective growth, but this decrease,particularly with the HUD205 transformants, was more marked than with any of the Ddp2 constructs described above. After 160 generations of nonselective growth less than 1% of the HUD205 transformant colonies still contained plasmid. We attribute the greater instability of pnDEAI to the deletion of an additional 630 bp of the native Ddp2 DNA lying 3’ to the Ddp2 rep ORF.

mant) and in HUD902 (+) and HUD91 1 (A) (AX4 transformants); (C) pnDAX4 in HUD906 (0) (HUD205 transformant) and in HUD901 (+) and HUD910 (A) (AX4 transformants); and (D) p60 in HUD77 I (+), HUD857 (a), and HUD858 (m) (AX4 transformants). The x-axis indicates the number of generations the cells were grown in the absenceof G4 18, and the y-axis indicates the ratio of the number of colonies able to form fruiting bodies in the population on medium containing G4 18divided by the number of colonies able to form fruiting bodies in the population on medium lacking G418.

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The basis of the vector maintenance assay is a difference in developmental phenotype which is dependent on the presence or absenceof a functional G4 18 resistance gene on the vector DNA. The assumption with the assay is that loss of the G418 resistance trait indicates loss of the vector. To test this we examined 100 AGG- colonies obtained from the experiments described above. DNA hybridization indicated that 88 of these lacked detectable plasmids, while the remaining 12 contained plasmids which appeared to be derived by deletion of vector sequences.As expected, the G4 18 resistance gene was affected in each of three independent deletions examined more fully. Thus the vector maintenance assay provided a good measure of the loss of vector DNA. One parameter that could be influenced by these constructs is the copy number of the vector DNA in the transformants. We screened for effectson copy number with the highly stable vectors p7d2, p27d2, and p3 ld2 and the lessstable p7 1d2 vector. These experiments were done with cells grown in axenic medium at 0, 10, and 20 pg G4 18/ml. Vectors p7d2 and p27d2 both had a high plasmid copy number which was uninfluenced by the presence or absence of G418 in the medium (150/tell and 1OO/cell, respectively). In contrast, in the absence of G4 I8 the copy numbers of p3 ld2 and p7 ld2 were lower (50 or fewer/cell) and in the presence of G4 18 their copy numbers increased in response to the selective pressure. Thus the deletion of one element of the Ddp2 inverted repeat had only a small effect on vector copy number while the presence of bacterial plasmid DNA had a much greater effect. However, reduced copy number was not directly correlated with plasmid instability, since the low copy number plasmid p3 1d2 was stable.

characteristics of integrative vectors, we analyzed three vectors derived from Ddp2: pnDdp2, pnDHI1, and pnDAX4 (Leiting et al., 1990). Each carries a different disrupted version of the rep gene in addition to having the G418 resistance gene cassette and the pUC19 bacterial vector. We also examined ~60, a plasmid containing only the G4 18 resistance gene cassette in pGEM3Z. In each case, the vectors were integrated in tandem arrays of 100 or more copies in the transformant DNA. Overall with these vectors there was greater variation in the stability of transformants derived from the same recipient strain than was seenwith autonomously replicating vectors (Fig. 3). A second feature of the results was the absenceof a strain-dependent difference in vector maintenance with the integrating vectors. Transformants derived from HUD205 were not consistently less stable than ones derived from AX4. The complete sequenceof Ddp2 and analyses of plasmid elements required for extrachromosomal replication have recently been published (Chang et al., 1990; Leiting et al., 1990). In particular, Leiting et al. (1990) described two plasmid constructs exhibiting very interesting phenotypes: pnDEI1, which lacks almost all of both elements of the inverted repeat region and appeared capable of low copy number autonomous replication, and pDA27a, which lacks the 3’ end of the Ddp2 rep gene and appeared to replicate autonomously at a high copy number but was lost or integrated after more than 60 generations of growth without selection. We obtained both constructs from Dr. A. Noegel to examine them using our maintenance assay. Plasmid pnDEI1 was transformed into strains AX4 and HUD205. Significantly, the HUD205 transformation frequency was about 1 in 2 X lo6 cells, a factor of 10 below that seenwith AX4 recipient cells. As shown in Fig. 4, maintenance data obtained with Ddp2 SequencesEssentialfor Autonomous pnDEI1 transformants were very different Replication from that obtained with transformants carryTo confirm that disruption of the rep gene ing pnDEA1 and the other Ddp2 vectors capaprevents autonomous replication ofDdp2-de- ble of autonomous replication (Fig. 2). The rived vectors and to study the maintenance pnDEI1 maintenance data closely resembled

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A 1

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FIG. 4. Maintenance of pnDEI1 and p103d2: (A) pnDEI1 in HUD205 transformants HUD904 (0). HUD915 (O), HUD916 (W),HUD938 (+), and HUD939 (a);(B) pnDEII in AX4 transformants HUD898 (+), HUD899 (a), HUD908 (w), HUD9 13 (V), HUD914 (-), HUD936 (A), and HUD937 (x); (C) p103d2 in HUD205 transformant HUD930 (0) and in AX4 transformants HUD929 (+), HUD93 I (A), HUD932 (A), and HUD933 (m). The x-axis indicates the number ofgenerations the cells were grown in the absence of G418, and the y-axis indicates the ratio of the number of colonies able to form fruiting bodies in the population on medium containing G418 divided by the number of colonies able to form fruiting bodies in the population on medium lacking G4 18.

that of known integrating vectors (Fig. 3). There were large differences in stability among the transformants with some being very stable but others very unstable. Like the integrating vectors, pnDEI1 did not show a greater stability in AX4 transformants than in HUD205 transformants. Therefore, on the basis of the transformation frequency in HUD205 and the maintenance assay, pnDEI1 appears to be an integrating vector incapable of autonomous replication. To confirm this, we analyzed DNA from pnDEI1 transformants on pulsed-field gels and compared the results to those obtained with known integrating and extrachromosomally

replicating vectors. Hybridization to integrating vectors and to pnDEI1 was distributed in fragments of 100 to 300 kb, the same as total nuclear DNA on the ethidium bromide-stained gel (Fig. 5). The distribution pattern obtained with the integrating vectors is exactly the result expected for these DNAs, since we know from restriction digests that they were present in tandem arrays of about 100 copies, which should be sheared at random sites in the preparation of the nuclear DNAs. In contrast, extrachromosomally replicating vectors produced discrete bands running at the same positions in preparations of total nuclear DNA as the bands in prepara-

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A similar analysis was done with a derivative of plasmid pDA27a, which is missing the 3’ end of the rep gene, one element of the inverted repeat, and all of the region between the 3’ end of the rep gene and the inverted repeat. We inserted a G4 18 resistance gene cassette between the Ddp2 component and the bacterial vector of pDA27a to give plasmid p103d2 (Fig. 1). Vector p103d2 is indistinguishable from known integrating vectors in the maintenance assayand on pulsed-field gels (Figs. 4 and 5). Vector p103d2 transformed HUD205 less well than it did AX4. Its copy number was similar to that of the integrating vectors at about 100 copies per cell. We therefore concluded that neither pnDEI1 nor p 103d2 was capable of autonomous replication.

Maintenance of a Ddpl Extrachromosomal Vector FIG. 5. Pulsed-field gel electrophoresis of total nuclear DNA samples from plasmid-free recipient strain AX4 (lane 2); from transformants containing pnDEA1: HUD935 (lane 3) HUD934 (lane 4); pnDEI1: HUD913 (lane 5) HUD898 (lane 6); p103d2: HUD929 (lane 7) HUD931 (lane 8); pnDAX4: HUD910 (lane 9) HUD901 (lane 10); Ddp2: HUD942 (lane 11); p7d2: HUD378 (lane 12). Purified plasmid DNA samples Ddp2 (lane 13), p7d2 (lane 14) and pnDEA1 (lane 15). Lane 1 contains bacteriophage X DNA digested with restriction enzymes EcoRI and HindIII: the largest linear DNA fragment is 2 1 kb. (A) Ethidium bromide-stained gel. (B) A Southern blot of the gel shown in (A) hybridized with a digoxigenin-labeled DNA fragment containing most of the Ddp2 open reading frame. The autonomously replicating plasmids pnDEA1, Ddp2, and p7d2 are detectable as discrete bands by hybridization in the nuclear DNA samples and run at the same positions on the gel as the purified plasmid DNA samples.

tions of purified vector DNA. This is the expected result with these relatively small (x 10 kb), extrachromosomal plasmids which should not be extensively sheared during the preparation of the DNA. Conventional electrophoresis of restriction enzyme-digested DNA from the pnDEI1 transformants suggestedthat the plasmid had a copy number of about 100 per cell.

We also analyzed the maintenance of p88d1, a shuttle vector that contains a 2.0-kb region of Ddp 1 carrying the Ddp 1 origin of replication, pGEM3Z, and a G4 18 gene cassette, but does not contain any functional Ddpl genes. In contrast to the extrachromosomal Ddp2-based vectors described above, p88dl was very unstable in transformants derived from either HUD205 or AX4. By 60 generations of nonselective growth, all six independently derived p88d 1 transformants analyzed were cured of p88d 1. No AGG+ colonies were seen. This result clearly indicates that p88d 1 lacks Ddp 1 regions that are essential for vector maintenance under nonselective growth. Vector p88dl can be maintained only by growth in the presenceof G4 18. Thus additional native Ddp 1 plasmid sequences must play a major role in maintaining Ddp 1 in D. discoideum cells. DISCUSSION

The selection and growth of transformants on agar medium with live bacteria allow the rapid isolation and visual screening of the developmental phenotypes of large numbers of independently derived colonies and their

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analysis under continuous selective pressure. This will be of great benefit, for example, in screening transformants for targeted modification of developmentally important genes and in studies involving gene isolation by complementation. Our lab is particularly interested in examining the mechanisms that D. discoideum plasmids have evolved to ensure stable maintenance in the cell nucleus. Such mechanisms are critical to avoid the formation of plasmidfree cells, which we showed have a growth advantage over cells bearing plasmids. These studies require that we can distinguish whether a particular vector construct exists extrachromosomally or is integrated into the chromosome and, if extrachromosomal, follow the appearance of plasmid-free cells in the population under nonselective conditions. The assayswe describe here allow a rigorous examination of these properties. Our findings with vectors derived from Ddp2 are summarized in Table 1. Vector p7d2 which carries the entire Ddp2 sequence (Fig. 1) was highly stable and almost never lost from either AX4- or HUD205derived transformants. However, removal of one element of the inverted repeat (p27d2) or insertion of a bacterial plasmid to construct a shuttle vector (p3 ld2) led to small decreases in plasmid stability. In contrast, when these two modifications occurred together they had a substantial effect (p30d2, p71d2, p86d2, and p87d2), such that in the HUD205 transformants after 160 generations in the absence of selection one half of the cells in such populations were typically plasmid-free. Deletion of an additional 630 bp of Ddp2 sequence in the region between the inverted repeat and the 3’ end of the rep ORF resulted in an even more rapid loss of vector DNA (pnDEA1). Since this deletion leaves the rep ORF and its polyadenylation signal intact, the simplest explanation of our data is that a sequence element in the deleted region is involved in plasmid maintenance. Analyses of vectors in which the rep gene was disrupted (pnDdp2, pnDHI1, pnDAX4, and p103d2) confirmed that an intact rep gene was re-

“ECTOR

MAINTENANCE

57

quired for extrachromosomal maintenance. Similarly, deletion of most of both elements of the inverted repeat (pnDEI1) led to the inability to replicate autonomously. We were unable to confirm the findings of Leiting et al. (1990) that pnDEI1 and pDA27a were capable of autonomous replication (Figs. 4 and 5, Table 1). The native plasmid Ddp2 contains three copies of a 49-bp element within the putative origin region spanning the inverted repeat and the sequence 5’ to the rep gene. Chang et al. ( 1990) concluded from their deletion studies that two copies of this 49-bp sequencewere essential for extrachromosomal replication. Leiting et al. ( 1990), based on data from pnDEI1, concluded that a single copy of the 49-bp repeat was sufficient for extrachromosomal replication. Our data with pnDEI1 are consistent with that of Chang and co-workers. Experiments with pDA27a led Leiting et al. (1990) to conclude that deletion of 174 bp from the 3’ end of the rep ORF allowed extrachromosomal replication at high copy number for a brief period of time. Our data with a derivative of pDA27a containing the G418 resistance gene (p103d2) showed no evidence for extrachromosomal replication. This result is not surprising, since Leiting and co-workers described pDA27a as being sometimes integrated into chromosomal DNA and very unstably maintained as an extrachromosomal vector. Two major differences between recipient strains HUD205 and AX4 were noted in this work. First, there is the difference in the ability to be transformed by integrating vectors. The lower transformation frequency of HUD205 than of AX4 with integrating vectors was unexpected and remains to be further characterized. Second, autonomously replicating vectors are less stable in HUD205 than in AX4. This was not unexpected but also remains to be explained. HUD205 was isolated as a strain that had spontaneously lost the Ddpl plasmid (Jensen et al., 1989; Hughes and Welker, 1989), and we specifically chose HUD205 as a strain that was apt to confer decreased vector stability. The in-

58

HUGHES, PODGORSKI, AND WELKER TABLE 1 FEATURESOFDdp2-DERIVED VECTORS

Vector

Extrachromosomal vs integrated

Vector stability”

Transformation efficiency*

pld2 p27d2 p30d2 p31d2 p7ld2 p86d2 p87d2 pnDEA1 pnDEI1 pnDdp2 pnDHI1 pnDAX4 pl03d2 ~60

E E E E E E E E I I I I I I

High High Intermediate High Intermediate Intermediate Intermediate Low Variable Nd NDf NDf Variable NDf

High High High High High High High High Low Low Low Low Low Low

Copies of Copies of 0.5-kb 49-bp Intact Intact 3’ Bacterial IR’ DRd ORF sequence< plasmid 2 1 1 2 1 1 1 1 0 2 1 1 1 0

3 2 2 3 2 2 2 2 1 3 2 2 2 0

+ + + + + + + + + -

+ + + + + + + + -

+ + + + + + + + + + + +

a Based on the percentage of HUD205 transformants containing vector after 160 generations without selection: high > 95%; intermediate = approximately 50%, low < 1%. ’ Using HUD205 as a recipient: high = at least IO-’ to 10m6,low = 10m6or less. ‘The native Ddp2 plasmid contains an inverted repeat composed of two 0.5-kb elements. d The 49-bp direct repeat identified by Leiting et al. (1990) and Chang et al. (1990) in putative origin region of Ddp2. Native plasmid has three copies spread through the inverted repeat and adjacent area 5’ to the rep gene. ‘Vector carries the sequence found 3’ of the 2.7-kb rep ORF between the gene and the inverted repeat region. IToo few HUD205 transformants containing these vectors were analyzed, but based on AX4 transformants analyzed (Fig. 3) we predict variable maintenance of these vectors in HUD205 transformants.

creased vector loss in HUD205 is thought to be a heritable trait, since related strains also showed loss of a native plasmid DNA (Hughes and Welker, 1989). We identified four features that consistently distinguished integrating vectors from those capable of autonomous extrachromosomal replication: (1) the decreasedability of integrating vectors to transform HUD205; (2) the greater variation in the maintenance of integrating vectors in different transformant clones; (3) while autonomously replicating vectors were less stable in HUD205 than in AX4, integrating vectors were not; and (4) the presence of the plasmid DNA in a broad distribution of DNA fragment sizes on pulsed-field gels compared to the discrete bands produced by small autonomously replicating vectors. The development of the vector maintenance assay and identification of several

traits consistently associated with integrating vectors will be useful in further analyses of the functions of native plasmid genesand sequence elements. We are particularly interested in those genesand elements involved in plasmid maintenance. The vector maintenance assay allows a much more definitive assessmentof vector stability than previously used protocols. For example, vectors carrying the Ddpl origin of replication but no functional Ddpl genes were claimed to be stable if, after several generations of nonselective growth, G4 1g-resistant cells could be recovered (Ahern et al., 1988). Yet p88d1, which carries just the Ddpl origin of replication, was the most unstable of all the vectors that were analyzed. This leads us to the markedly different conclusion that additional Ddp 1 sequences and/or gene products play a major role in plasmid stability, although they are not essential for replication of the plasmid.

TRANSFORMATION

PIND VECTOR MAINTENANCE

59

Clearly, two approaches using the maintelectable marker in L)ic~~~~stc/im~ di~c01~/~~117. llol Cell. Biol. 9, 1965-1968. nance assayare possible. In the present work vectors derived from Ddp2 and Ddpl that FARRER.N. A.. LONGHURST,T. J., LEIGH, D. A.. AND WILLIAMS,K. L. (1988). Nucleotide sequence of a dehave a specific gene or element inactivated velopmentally regulated Dict~wtelium discoidezo~enwere studied and compared to other vectors dogenous plasmid gene and 5’ flanking region. h’hjl&i(~ without these mutations. In future work it Acids Res. 16, 10.914. will be possible to take vectors such as p88d 1 GURNIAK, C. B., BANG. A. G., AND NOEGEL, .A. A. (1990). Transcript and sequence analysis of a 5. I kb that carry functional origins of replication contiguous fragment of Dict.wstehm discoidewn but are extremely unstable and add to them plasmid Ddpl that contains the origin of replication specific plasmid sequences.This should proand codes for several transcripts. Curr. Gmet. 17,32 I vide an easy way to identify genes or se325. quence elements that play a role in plasmid HOWARD, P. K., AHERN. K. G., AND FIRTEL. R. A. (1988). Establishment of a transient expression system maintenance by the increased stability of the for Dictyostelium discoideum. Nucleic Acids Rex 16, vectors carrying them. 2613-2623.

ACKNOWLEDGMENTS We thank Dr. Angelika Noegel for her gifts of vector DNA. We also thank C. Peterson for typing this manuscript. This work was supported by NSF Grant DMB8915591.

REFERENCES AHERN, K. G., HOWARD, P. K., AND FIRTEL, R. A. ( 1988).Identification ofregions essential forextrachromosomal replication and maintenance of an endogenous plasmid in Dictyostelium. Nucleic Acids Rex 16, 6825-6837.

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HUGHES,J. E., AND WELKER,D. L. (1989). Copy number control and compatibility of nuclear plasmids in Dictyostelium discoideum. Plasmid 22, 2 15-223. HUGHES,J. E., ASHKTORAB,H.. AND WELKER, D. L. (1988). Nuclear plasmids in the Dictyostelium slime molds. Der. Genet. 9,495-504. JENSEN,S. L.. ASHKTORAB,H.. HUGHES, J. E., AND WELKER,D. L. (1989). Gene amplification associated with the dominant cob-354 cobalt resistance trait in Dictyostelium discoideum. Mol. Gen. Genet. 220, 25^^ 3L. KALPAXIS, D.. WERNER,H.. BOY-MARCOTTE,E., JACQUET,M., AND DINGERMANN,T. (1990). Positive selection for Dictyostelium mutants lacking uridine monophosphate synthase activity based on resistance to 5-fluoroorotic acid. Dev. Genet. 11, 396-402. KNECHT, D. A., JUNG, J., AND MATTHEWS,L. (1990). Quantitation of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells. Dev. Genet. II, 403-409. LEITING, B., LINDNER,I. J., AND NOEGEL,A. A. ( 1990). The extrachromosomal replication of Dicryostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor. Mol. Cell. Biol. 7, 3727-3736.

METZ, B. A., WARD, T. E., WELKER,D. L., AND WILLIAMS, K. L. (1983). Identification of an endogenous plasmid in Dictyostelium discoideum. EMBO J. 2, 515-519. NELLEN, W., SILAN, C., AND FIRTEL, R. A. (1984). DNA-mediated transformation in Dictyostelium discoideum: Regulated expression of an actin gene fusion. Mol. Cell. Biol. 4, 2890-2898. NOEGEL,A., WELKER, D. L., METZ, B. A., AND WILLIAMS, K. L. (I 985). Presence of nuclear associated plasmids in the lower eukaryote Dictyostelium discoideum. J. Mol. Biol. 185,441-450.

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