Optimization and in situ detection of Escherichia coli β-galactosidase gene expression in...

Gene, 85 (1989) 353-362 Elsevier

353

GENE 03339

Optimization and in situ detection of Escherichia coli b-galactosidase gene expression in Dictyostelium discoideum

(Recombinant DNA; genetic transformation; heterologous IacZ expression; gene fusion; D19 promoter; actin 6 promoter; transcription termination; slime molds)

Theodor Dingermann a) Norbert Reindl p, Herbert Werner *, Martin Hildebrandt p*, Wolfgang Nellen b, Adrian Harwood’, Jeffrey Williams’ and Kithe Nerke”

n Institut ftir Biochemie der Medizinischen Fakultiit, D-8520 Erlangen (F.R. G.). b Max Planck Institut ftir Biochemie, Abt. Zellbiologie, D-8033 Martiwied (F.R. G.) and’ Imperial Cancer Research Fund, Glare Hall Laboratories, South Mimms, Herts, EN6 3LD (U.K.) Tel. (0707)44444

Received by K.F. Chater: 25 July 1989 Revised: 1 September 1989 Accepted: 5 September 1989

SUMMARY

We show that a fusion gene, containing the promoter and 5’noncoding region of a Dictyostelium discoideum

actin 6 gene linked to the Escherichia coli /I-galactosidase (/IGal) gene (lacz), directs the production of functionally active j?Ga.l in D. discoideum and that the enzyme can be detected by staining in situ; a procedure which will be of great value in analyzing cell-type-specific gene expression. We illustrate this by fusing ZacZ

to the promoter of the prespore-specific gene, 019, and localizing expressing cells in migrating slugs. Optimal expression requires the inclusion of termination and polyadenylylation signals and we describe pDDlac, a vector containing a multiple cloning site upstream from a lacZ-Dictyostelium terminator fusion, which can be used to analyze regulated promoters.

INTRODUCTION

Gene and operon fusions to the E. coli IacZ gene encoding #IGal (EC3.2.1.23) have proven to be

Correspondence to: Dr. T. Dingermann, Institut Rlr Biochemie der Medizinischen Fakultat, Universitat Erlangen-NUmberg, Fahrstrasse 17, D-8520 Erlangen (F.R.G.) Tel. (09131)854189; Fax (09131)852131. * Present address: Max Planck Institut Blr Biochemie, Abteilung Zellbiologie, D-8033 Martinsried (F.R.G.), Tel. (089)85782329.

Abbreviations: Ap, ampicillin; /IGal, B-galactosidase; bp, base pair(s); CPRG, chlorophenolred-B-D-galactopyranoside; D.,

Dictyosrelium; DMSO, diiethyl sulfoxide; EGTA, ethylene glycol-bis(/I-aminoethyl ether) tetraacetic acid; G418, amino-

powerful tools in prokaryotic molecular genetics (see Bassford et al., 1978; Silhavy et al., 1984). Analo- gous constructs, producing functional j?Gal under transcriptional control of a eukaryotic promoter, are

glycoside antibiotic G418; kat, see Table I, footnote d; kb, kilo- base(s) or 1000 bp; MCS, multiple cloning site; neo, neomycin- resistance gene; nt, nucleotide(s); ONPG, o-nitrophenyl- p-D-galactoside; on’, origin of DNA replication; PAGE, polyacrylamide-gel electrophoresis; PBS, 70 mM Na,HP0,/30 mM KH,PO,/lSO mM NaCl; PUE, positive upstream element; R, resistant; SDS, sodium dodecyl sulfate; SSC, 150mM NaCl/lS mM Na, . citrate pH 7.6; TPBS, see MATERIALS AND METHODS, section c; UAS, upstream activator sequence; XGal, 5-bromo-4-chloro-3-indolyl-/I-D-galactoside; Z-buffer, see MATERIALS AND METHODS, section C.

0378-l 119/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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similarly useful to study gene expression in eukaryotes (Rose et al., 1981; Guarente and Ptashne, 1981; Lis et al., 1983; Teeri et al., 1989).

D. discoideum is a simple system in which to study the mechanisms controlling development (see Loomis, 1982, for review). Upon starvation individual amoebae aggregate to form a multicellular structure which differentiates to form a fruiting body con- sisting of spores supported by dead, vacuolated stalk cells. A migratory slug stage may form as an inter- mediate to this final structure, containing prestalk cells in its anterior fifth and prespore cells in its posterior four-fifths. Large numbers of stage and cell-type-specific genes have been isolated (Williams et al., 1979; 1985; Rdwekamp and Firtel, 1980; Barklis and Lodish, 1983; Mehdy et al., 1983; Datta et al., 1986; Noegel et al., 1986).

The ability to create la&? gene fusions in D. discoideum would be of value. Such constructs would allow the assay of developmentally regulated promoters by staining in situ. The assay of /?Ga.l is technically less demanding than immunohistochem- istry and can be equally sensitive.

We have fused parts of two different D. discoideum

actin genes to the E. coli 1acZ gene to create a system which allows the detection of /IGal activity in D. discoideum. Actin 6 is one of 17 members of a multigene family in D. discoideum (McKeown et al., 1978 ; McKeown and Firtel, 198 1 a,b). The mRNA is preferentially expressed in growing cells. Shortly after the onset of development actin 6 message gradually decreases to comprise about 2% of the actin mRNA population at culmination (Kimmel and Firtel, 1982). Actin 8 is expressed during growth at a slightly lower level than the actin 6 gene but whose expression remains high throughout development (Kimmel and Firtel, 1982).

A third gene used in these studies, D19, is first expressed after 10 h of development and it is maxi- mally expressed in the slug stage, where its mRNA is at least tenfold more abundant in prespore cells than prestalk cells (Barklis and Lodish, 1984; Krefft et al., 1984). The DI9 gene has been cloned (Early et al., 1988) and by expressing the IacZgene from the Dl9 promoter, we show that j?Ga.l activity can be visualized in prespore cells.

MATERIALS AND METHODS

(a) Strains, media and Dictyostelium discoideum

transformation

Axenic strains Ax-2 and AX-~, obtained from G. Gerisch, were grown in HL-5 medium (Watts and Ashworth, 1970) at 22°C. Cells ‘used for transformation were grown in morpholine-ethanesulfonic acid-buffered HL-5 medium (Nellen et al., 1984). D. discoideum transformants were obtained as described (Nellen et al., 1984; 1986; Nellen and Firtel, 1985; Early and Williams, 1987) using strains Ax-2 or KAx-3 (Poole and Firtel, 1984). Routinely, 5 pg of plasmid DNA, containing the fusion gene, was co-transformed with 5 pg of the G418 resistance vectors pBlOSX (Nellen et al., 1984), pNeoI-T or pDNeo-2 (Knecht et al., 1986). Transformants were selected in the presence of 20 pg/ml G4 18. Two days after transformation, floating cells were carefully washed off the petri dish with a Pasteur pipette and the G418 concentration was lowered to 5 pg/ml. Washing and replacement of media were repeated at one- to two-day intervals resulting in a loss of most of the nonattached (nontransformed) cells. Clonal growth of the adherent transformants was apparent four to live days after the transformation. Primary colonies were picked from the petri dish under a dissecting microscope using a sterile drawn-out Pasteur pipette and transferred to microtitre wells containing 5 c(g G418/ml. Depending on the cell density on the transformation plate, these clones were usually pure. In most cases they were, however, recloned on bacterial lawns. Southern analysis was performed on genomic DNA isolated from transformants. As expected, these analyses showed them to contain multiple copies of, apparently tandemly integrated, vector and fusion gene molecules (Nellen et al., 1984).

(b) Northern analysis

Total RNA was prepared from 5 x lo’-1 x lo* washed cells. Cells were lysed in 3.5 ml 4 M guanidium isothiocyanate and quickly drawn several times through a syringe fitted with a needle until the

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viscosity of the solution decreased. The RNA was pelleted through a cushion of 1.5 ml of 4.7 M CsCl (35000 rev./nun, 18°C 16 h), dissolved in 360 ~1 TES buffer (10 mM Tris * HCl pH 7.4/5 mM EDTA/l% SDS) and precipitated with 2.5 ~01s. ethanol at -80°C. Purified RNA was stored in sterile water at -80°C.

RNA was separated by formaldehyde gel electrophoresis (Lehrach et al., 1977) in 1.2% agarose gels, immobilized on GeneScreen plus membranes and hybridised with nick-translated DNA fragments. Hybridisations were incubated at 42’ C in 50% form- amide, 4 x SSC, 1 y0 SDS, and 1 x Denhardt’s solution. After hybridization filters were washed 3 x 10 min in 2 x SSC at room temperature, 2 x 30minin2 x SSCplusO.5% SDSat65”Cand 1 x 20 min in 0.1 x SSC at room temperature.

(c) Enzyme assays for /IGal

D. discoideum cells were harvested by centrifuga- tion (500 x g, 5 min) and washed twice in phosphate buffer (14.7 mM KH,P0,/2 mM Na,HPO, pH 6). Assays were performed by the standard method (Miller, 1972) with slight modifications. Cells (5 x 106-5 x 107) were suspended in 1 ml phosphate buffer. After a freeze/thaw cycle, the sus- pension was vortexed for 1 min and cleared by cen- trifugation for 5 min in an eppendorfcentrifuge. Two ~1 of extract were used to determine protein concen- trations by the dye-binding method (Bio-Rad). /IGal activity was assayed from 100 ~1 extract. 300 ~1 Z-buffer (60 mM NazHP0,/40 mM NaH,PO,/ 10 mM KCl/l mM MgSO, pH 7), 50 mM /I-mercaptoethanol and 200 ~1 of a solution containing 4 mg/ml ONPG in 100 mM phosphate buffer pH 7 were added and assays were incubated at 22°C. Reactions were stopped by adding 400 ~1 of 1 M Na,CO, and cleared for 5 min in a microcentrifuge. An aliquot of 500 ~1 was diluted with 500 ~1 Z-buffer and the absorbance was determined at 420 nm. Specific enzyme activities are given as kat/mg protein. One kat is defined as the enzymatic activity which hydrolyses 1 mol of ONPG/s at 22°C.

Alternatively 5 x 105-1 x lo7 lysed cells were transferred to microtitre dishes which were precoated with anti$Gal antiserum. After 4-16 h at 4°C the wells were rinsed three times with TPBS buffer (100 mM KH,PO, + Na,HPO, pH 7.2/

150 mM NaCl/O. 1% Tween-20) and 300 ~1 of substrate solution (0.4 mg CPRG/ml/lOO mM Hepes pH 7.5/10 mM MgCl,/lSO mM NaCl/O.OS % Tween-20/28 mM /I-mercaptoethanol) was added to each well. Aliquots were withdrawn at various times of incubation and absorbance was measured at 576 nm.

Dictyostelium cells express an endogenous j?Gal activity (Dimond et al., 1976) and the activity is prestalk-enriched (Oohata and Takeuchi, 1977). However, the Dictyostelium enzymes are active only at acid pH (Dimond et al., 1976) and we never experienced any background problems with untransformed control cells.

(d) Detection of /.?Gal by SDS-PAGE

/IGal can be detected by SDS-PAGE (Teeri et al., 1989). Extracts corresponding to 40 pg of protein were mixed with cold loading buffer and applied directly to an 8% SDS-polyacrylamide gel (Laemmli, 1970). After electrophoresis at 4°C the separation gel was washed once in cold Z-buffer and twice in Z-buffer for 15 min at room temperature. The substrate 4-methyl-umbelliferyl-~-D-galactopyranoside was dissolved at 20 mg/ml in DMSO and diluted lOOO-fold in Z-buffer containing 50 mM /?-mercaptoethanol. The gel was agitated for 10 min in the substrate solution at room temperature, rinsed with water and fluorescent enzyme bands were photographed under UV illumination through a pale yellow filter (Kodak Wratten 2E).

(e) Histochemical staining

Cells were applied to poly-lysine precoated slides and were allowed to settle for 15 min. After removal of medium, cells were incubated in fixing solution (1% glutaraldehyde, 1 mM EGTA in Z buffer) for 30 to 60 min. Slides were washed twice in Z-buffer and the cells were overlaid with 200-400 ~1 staining solution (50 ~1 100 mM K,[Fe(CN),]/50 ~1 100 mM Kq[Fe(CN)6]/50 ~120 mM XGal in N,N- dimethylformamide/100 ~1 10 mM EGTA/750 ~1 Z-buffer). After incubation for 5 to 15 h at 37” C in a moist chamber, cells were washed twice in Z-buffer and fHed in methanol for 5 to 10 min.

Multicellular stages of D. discoideum were developed on phosphate buffer-saturated nitrocellulose

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s.j NW In -

- 0.) DamIN cluvrgm

1 ~A6P14 11 n plrrlu I.c~~

iiTTTTTTTT TTTTTGAGGT TTCTGAGATj..iii@GAA ATTTTTTT'TT

TTTTTTTAAT TiATTCAAA4 AAATAATCAA ATAAATAAAT ATAATATAAi

ATG GAT GGT GAG GGA ATT CAT CGG GGC AAT TCA CTG GCC GTC

met asp gly glu gly ile his arg gly as" ser leu ala val

Fig. 1. The construction of pA6PTlac.l. The PUE of the actin 6 promoter (Nellen et al., 1986) was isolated as a FspI fragment from pBlOSX (Nellen and Firtel, 1985) and ligated into the SmaI site of pUC18. After digestion with EcoRI, end-filling and BamHI digestion the PUE was ligated into the end-filled Sal1 site-unmodified BumHI site of pIC20A8T. The resulting plasmid was termed pA6P*A8T. The promoter was isolated as a FspI-BulI fragment from pBlOSX and was cloned into the SmaI site of pUC9. After linearization with EcoRI and limited BAL 31 digestion, EcoRI linkers were added and fragments were liberated with HindIII. Protruding ends were filled

filters which were placed on 2% phosphate agar. Filters were incubated in fixing solution for 30 min, washed twice in Z-buffer and overlayed with staining solution. After incubation at 37°C filters were washed twice in Z-buffer and photographed.

RESULTS AND DISCUSSION

(a) Fusion constructs of act6: : lucZ

The actin 6 promoter is comprised of a PUE, between positions -599 and -572, and an UAS between positions -249 and -215 (Nellen et al., 1986). A fragment containing these elements was fused to the 1ucZ gene at a point just downstream from the actin start codon (Fig. 1). Most of the actin 6 N-terminal coding nt were deleted with BAL 31 exonuclease. The resulting fusion gene contains only three actin 6 codons and live codons introduced by the MCS (Fig. 1). A transcriptional termination/ polyadenylation signal was inserted downstream from the 1ucZ coding region. This comprises two tandemly organized actin 8 termination/polyadenylylation signals isolated from plasmid pIC20A8T (kindly provided by A. Noegel). The resultant plasmid, pA6PTlac.1, was co-transfected into D. dis-

coideum with pNeol-T (Knecht et al., 1986). This latter plasmid contains the neoR gene from Tn903 fused to the actin 15 promoter and transcription terminator. The G418-resistant clones, A6PTlac1/6 and A6PTlac1/7, were isolated and shown to contain the fusion gene (data not shown).

To assess the importance of the actin 8 termination/polyadenylylation sequences for correct expression of the IacZ gene in D. discoideum, they were eliminated from pA6PTlac.l by digestion with Hind111 + XhoI, filling the protruding ends and recircularisation of the plasmid. The resultant plasmid, pA6Plac, was used to transform Ax-2 cells and two clones (A6Plac15/2 and A6Plac23/2) were iso-

lated.

a b

Fig. 2. Northern analysis of D. discoideum transformants. (Panel a) Northern analysis ofDictyostelium cells co-transformed with fusion gene plasmids. Whole cell RNA was prepared from Ax-2 cells and from individual cell-clones transformed with fusion gene plasmids. After formaldehyde gel electrophoresis (Lehrach et al., 1977) in 1.2% agarose gels the RNA was blotted to GeneScreen Plus@ membrane and hybridised with a nick- translated CZuI-XhoI fragment isolated from pIP210 (this fragment carries the C-terminal part of the ZacZ gene). Only in A6PTlac.l transformants (lanes b and c) were specific mRNAs of predicted size detectable. (Panel b) Northern analysis of total cellular RNAs isolated from A6PTlac1/7 at different developmental stages. Total cellular RNA was prepared from vegetatively growing cells (lane a) and from cells starved in phosphate buffer for 4 h (lane b) and 16 h (lane c). Separation of RNA and hybridisation conditions were as described above.

(b) Expression of the uct6: : ZucZ fusion gene

Total cellular RNA was prepared from individual transformed clones known to contain fusion gene copies and analysed by Northern blotting. Fusion gene transcripts were detected in the pA6PTlac.l transformants (Fig. 2a, lanes b and c) but were not detectable in the pA6lac transformants (Fig. 2a,

in and, alter digestion with EcoRI, the required fragment was ligated into the SmaI + EcoRI digested plasmid pA6P*A8T to yield pA6P14. This plasmid was finally used to insert the entire coding region of la&, present as a SmaI-XhoI fragment on pIP210. The resulting construct, pA6PTlac. 1, is approx. 7 kb long and contains an acti: : 1acZ fusion gene with only three actin 6 codons plus five codons introduced by the MCS. A, Asp718; B, BomHI; Bl, BaZI; C, C/al; E, EcoRI; F, FspI; H, HindHI; S, SalI; Sm, SmaI; X, XhoI; Xb, XbaI.

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TABLE I

Specific 8Cial activity in Dictyostelium dircoideum transformants

Clone a Terminator Specific activityd

A6PTlac1/7’ A6PTlacl/6b A6Plac23/2’ A6Plac15/2”

actin 8 actin 8

0.520 f 0.018 0.312 f 0.015 0.025 f 0.005 0.030 + 0.005

a DNA (5 pg) of the fusion gene was co-transformed with 5 pg of the selection plasmid (pNeol-T) and individual clones selected. All plasmids carried the actin 6 promoter. b pA6PTlac.l transformants. c pA6Plac transformants. pA6Plac is derived from pA6PTlac. 1 by deletion of the actin 8 terminator. d Cells were lysed by a freeze/thaw cycle and the extracts were tested for BGal activity and protein concentration. Specific activities are given as nkat/mg protein. 1 kat is defined as the catalytic activity which hydrolyses 1 mol of substrate (ONPG)/s at 22°C. Mean values were calculated from at least four individual tests.

lanes d and e). This demonstrates that the acf6: : IacZ gene fusion is transcribed to give functional IacZ mRNA in D. discoideum cells and suggests that the presence of a transcription termination/polyadenylylation signal is necessary to stabi- lize the IacZ transcripts.

Northern analysis was also used to investigate the pattern of expression of the act6 : : IacZ fusion gene in clone A6PTlac1/7 during development (Fig. 2b). The IacZ mRNA was highest in vegetatively growing cells. It declined shortly after the onset of development and in cells which had been starved for 16 h in phosphate buffer the mRNA was barely detectable. This pattern of expression of the actin 6 fusion gene mirrors that of the endogenous gene (Nellen et al., 1984; Knecht et al., 1986).

To determine whether the mRNAs transcribed from the 1acZ fusion genes were translated to pro- duce functional fiGal protein, extracts from each clone were assayed for enzymatic activity (Table I). Clones A6PTlac l/6 and A6PTlac l/7, containing both promoter and terminator, produced about 20-fold the activity of clones A6Plac15/2 and A6Plac23/2, which contain only the promoter. This demonstrates that the act6 : : 1acZ fusion gene synthe- sises functional fiGal when introduced into D. discoideum and further supports the conclusion that a transcription termination signal is essential for high-level expression. There was variability between different transformant clones in the level of j?Gal

activity. This correlated well with the copy number of integrated, intact, fusion gene molecules (not shown).

(c) Alternative means of detection of /ICal in Dictyostelium discoideum

In the results described so far expression of the IacZ gene was demonstrated using enzymatic assays. fiGal may also be detected after SDS-PAGE (Teeri et al., 1989) or by direct histochemical staining of whole cells. We applied both these methods to our transformants.

(I) SDS-polyacrylamide gel electrophoresis

Fig. 3 shows that, after separation in SDS- polyacrylamide gels, /3Gal activity can be directly visualized in proteins extracted from our transformants. The apparent level correlated well with levels found in the standard assay (Table I). Interestingly, however, enzyme activity levels did not reflect the developmental pattern of transcription seen by

c\IcucDb . . . . V)corr rcV 0 0 vomc(I ZZ’FF

c?anrl.n xcowww a a a a a PGal abcdefgh

N--- pGa1

Fig. 3. In situ staining of 8Gal after SDS-PAGE. Crude protein extracts (4Opg protein) from individual transformants were loaded onto a 0.1% SDS-lo% polyacrylamide gel (Laemmli, 1970) and separated for 5 h at 4°C. The gel was processed as described in MATERIALS AND METHODS, section b. After hydrolysis of 4-methyl-umbelliferyl-p-o-galactoside by BGal the fluorescent product was detected by UV illumination. According to the values summarized in Table I, the following enzyme activities were loaded:(b) A6Plac15/2 = 1 pkat; (c) A6Plac23/2 = 1.2 pkat; (d) A6PTlac1/6 = 12 pkat; (e) A6PTlac1/7 = 21 pkat. In lanes f-h BGa.l standards were loaded corresponding to 7 pkat, 0.7 pkat and 0.07 pkat, respectively.

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Northern analysis. There was almost no ditference in /IGal activity between growing cells and cells which had been starved for 4 h or 16 h in phosphate buffer. In slugs significant, although slightly lower, enzyme activities were detected. This indicates that the fiGal protein is stable even under the conditions of high protein turn-over which pertain during development. This is surprising because the fusion protein is large and heterologous gene products tend to be rapidly degraded. However, similar results have been obtained with the neoR gene in D. discoideum. When

expressed from a developmentally regulated promoter, enzyme levels stay high, even after RNA levels have decreased significantly (T. May and W.N., unpublished).

(2) Histochemical staining

One of the great attractions of the IacZ gene is that, using XGal as a substrate for the enzyme, a blue precipitate is produced which can readily be detected in individual cells. We have slightly adapted methods used for /IGal detection in mammalian cells (J. Price, personal communication) and in plant cells (Teeri et al., 1989) and can detect blue staining in D. discoideum cells transformed with pA6PTlac.l (Plate I, Fig. 4). Individually growing cells containing the acd: : IacZ transformant are stained blue (Plate I, Fig. 4a) while control, nontransformed cells are colourless (Plate I, Fig. 4b). For reasons which are unclear, there is considerable variation in the extent of staining. Although the progeny of a single clone were analysed, there may be continual changes in the extent of amplification of the fusion gene. Hence the variable expression may reflect variation in copy number between individual cells. Altema- tively, it may indicate variation in the extent of cell permeabilisation, resulting in different amounts of the substrates entering each cell. However, additional efforts to enhance the degree of cell permeabilisation such as adding detergent or DMSO to the fixative did not improve the results.

(d) Developmentally regulated expression of the 1acZ gene

The staining procedure can also be applied to whole mounts of developmental stages, such as the mid-cuhninant in Fig. 4c. Because the actin genes are expressed in a relatively non cell-type specific

manner (for example, the actin 3A,B and C transcripts of Tsang et al. (1982) are present only in about a threefold higher concentration in prestalk cells than in prespore cells) the cuhninants expressing the fusion gene are uniformly stained (Fig. 4~). Therefore, to demonstrate the feasibility of visualiz- ing cell-type-specific patterns of gene expression during development, we coupled ZacZ to the promoter of the 019 prespore-specific gene. A DNA fragment, containing the D19 promoter (Early et al., 1988), fused to the TATA box, start codon and a small number of N-terminal codons of the actin 15 gene (A. Early and J. Williams, manuscript submitted), was joined to the lacZ-act8 terminator region through a short linking piece of DNA to ensure the correct reading frame (Fig. 5a). The resulting plasmid, pD19lac-2 was introduced into Ax-2 cells by co-transformation with pDNeo-2, and G418- resistant clones isolated. Fig. 6 (Plate I) shows a stained slug developed from cells of a transformant clone D 19lac24/ 1. As expected for expression of the D19 gene, the stained cells are highly enriched in the posterior, prespore region of the slug. Again, not all cells in the posterior part of the slug are stained to the same extent. Because we are analyzing whole mounts, inadequate access of the ferrous and ferric cyanide compounds included to fur the blue precipitate can be a problem. (J. Price, personal communication). However, we observed no difference between samples incubated in a high (20 mM) or a low (5 mM) concentration of these compounds (not shown), or between samples which were fmed in the presence or absence of an additional detergent.

(e) Construction of pDDlac

To construct a generally useful cloning vector we removed the D19 promoter from pD191ac-2 to pro- duce pDDlac- 1. This plasmid contains a small MCS inserted upstream from the ZacZ-act8 terminator (Fig. 5b).

(f) Conclusions

We have demonstrated that the E. coli IacZ gene can be efficiently expressed in Dictyostelium transformants under the control of Dictyostelium promoters, and constructed a ZacZ expression vector suitable for use in this organism. Several highly sensitive

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Plate I. Fig. 4. (upper panel) In situ staining of A6PTlac1/7 transformants. Cells were applied to slides precoated with poly-lysine and fixed in 1 y0 glutaraldehyde for 30 min. Staining solution (see MATERIALS AND METHODS, section e) was overlayed and slides were incubated for 16 h in a moist chamber at 37°C. After fixing for 10 min in methanol, slides were air-dried and cells photographed. Multicellular stages were developed on nitrocellulose filters. At different developmental stages filters were incubated in fixing solution for 30 min and cells were stained as described (A) Vegetative cells of the A6PTlac1/7 transformant; (B) vegetative untransformed Ax-2 cells; (C)A6PTlacl/7 cells at culmination.

Fig. 6. (lower panel) In situ staining of multicellular stages of D191ac24/1 transformants. Transformed cells were developed and at different developmental stages fixed and stained as described in MATERIALS AND METHODS, section e. (A) Slug stage showing staining localised to the prespore region (i.e., posterior 80%); (B) pre-cuhninant stage showing staining localised within the ‘spore head to a region that corresponds to the position of the spore cells.

361

A Xb Eg E Sm

TCT Cda TIC CTA tCT AhA TCT TGb ATC GGC AA1 BGd

Xb Bg B Sm

& Al;A TCT TGb ATC C& GGC AAT TCA CTG

99 m :: actlnU fualon promotor y-w.“” ’ LF37 A8T I actin& tormhtor

L-?&q w

Fig. 5. Schematic drawings ofpDl9Iac2 and pDDlac-1. (A) Plasmid pDl91ac-2 contains theXbaI-Asp718 fragment from pD19 BamCat ligated to the lucZ-act8 terminator gene fragment in pUC9. The XbaI-Asp718 fragment comprixes the upstream 019 promoter elements fused to the actin 15 TATA box and N-terminal sequences (Early and Williams, 1989). The correct reading frame was obtained by ligating the linker, GTACCCTATCTAGATCTTG into the Asp718 and BumHI sites of the resulting 019: :lacZ fusion. The plasmid pDl9lac-2 expresses a fusion of jGal preceded by a short N-terminal actin 15 polypeptide. The diagram illustrates the DNA sequence at the point of the gene fusion. (B) To construct pDDlac-1 the promoter was removed from pDl91ac-2 by Xbal digestion and the plasmid was recircularized with T4 DNA ligase. The resulting plasmid contains the lucZ-act8 terminator fusion preceded by an MCS (sequence indicated).

methods are available to detect the enzyme and we have shown to be applicable to Dictyostehn. This offers the possibility of using this gene as a reporter to analyse developmentally regulated gene transcription. The establishment of a histochemical staining procedure means that it is also possible easily to define spatially regulated patterns of gene expression.

schalek for sequencing. We are grateful to Drs. Helga and Walter Kersten for support and encouragement and to Dr. Jack Price and Dr. William Loomis for their advice. This work was supported by grants

from the Deutsche Forschungsgemeinschaft, Fonds der Chemischen Industrie and the Johannes und Frieda Marohn-Stilbtng to T.D.

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