Nucleic Acids Research, Vol. 19, No. 21 5857-5862

The actin

gene

promoter of Trypanosoma brucei

M.F.Ben Amar, D.Jefferies, A.Pays, N.Bakalaral, G.Kendall1 and E.Pays* Department of Molecular Biology, University of Brussels, 67 rue des Chevaux, B1640 Rhode St Genese and 11CP, avenue Hippocrate 75, B1200 Brussels, Belgium Received August 30, 1991; Revised and Accepted October 11, 1991 ABSTRACT The actin genes of Trypanosoma brucei are transcribed constitutively during the parasite life-cycle, by a polymerase sensitive to ca-amanitin (1). The start region of the actin gene transcription unit was mapped by virtue of the accumulation of promoter-proximal transcripts which occurs following moderate UV irradiation (2 - 6). This region, located about 4 kilobases upstream from the genes, was able to direct transient expression of the bacterial Chloramphenicol Acetyl Transferase (CAT) gene in both bloodstream and procyclic forms of the parasite. The essential region of the promoter was defined by deletion, and appeared to be within 600 bp upstream from the putative transcription start site. It does not share significant homology with the other trypanosome promoters described so far (VSG, procyclin, rDNA), which all direct a-amanitin resistant transcription. INTRODUCTION The upstream regions which control the transcription of trypanosome genes are difficult to identify, due mainly to transsplicing of the transcripts and the polycistronic nature of transcription (for a recent review, see 7). To date, only a few transcription promoters have been described in T. brucei, all for RNA polymerase(s) resistant to -amanitin (5,8 - 12). We have a

previously reported a method, using moderate levels of UV irradiation, which allows the crude mapping of transcription start sites in trypanosomes, due to the accumulation of promoterproximal transcripts (2-6). This method, previously applied to the VSG, rDNA and procyclin promoters, has now been extended to the actin genes, the transcription of which is both constitutive and sensitive to a-amanitin (1). The putative promoter was mapped to a region approximately 4 kb upstream from the actin genes. When cloned into a CAT vector, this region was able to direct expression of the reporter gene in both bloodstream and procyclic forms of T. brucei.

MATERIALS AND METHODS Trypanosomes Bloodstream trypanosomes of T. b. brucei strain EATRO 1125 isolated from rat blood at the peak of parasitaemia by centrifugation (see below). Procyclic forms of the same strain were

*

To whom correspondence should be addressed

GenBank accession

no.

M76487

25-27°C in SDM-79 (13), or Cunninghams medium (14), supplemented with 10-15 % inactivated foetal calf serum and 10 jig/ml gentamycin.

were grown at

DNA and RNA analysis The procedures for DNA and RNA isolation, Southern and Northern blot hybridization as well as DNA cloning, were as described (15). A 13 kb SalI fragment, containing the first copy of the tandemly-linked actin genes together with 11 kb of upstream sequence, was cloned from the AnTat 1.3A DNA of T. brucei by ligation with the Sall arms of the bacteriophage EMBL3 vector. For most analyses of the actin promoter region, a 2.6 kb HindIH fragment from the EMBL3 recombinant was subcloned into the plasmid PUC 18. The sequences of DNA fragments, subcloned in bacteriophage M13 derivatives, were determined on both strands by the method of Sanger et al. (16), using a modified T7 DNA polymerase (Sequenase, United States Biochemical Corp.).

Plasmids Plasmids were either prepared by the alkaline lysis method, and purified by caesium chloride density gradient centrifugation, or by the boiling lysis method for plasmid minipreps, using 10 ml of overnight culture (17). The actin promoter-dependent CAT vector (pDAV) was obtained by deleting the 1.1 kb BamHI-StuI fragment containing the VSG promoter and downstream region from the CAT expression plasmid pD5V (9), and replacing it by a 1.2 kb BamHI-HindlI fragment, thought to contain the actin promoter, after blunt-ending of the Hindl site. In plasmid pDAV as in pD5V, the 3' splice site in front of the CAT gene is that of ESAG 7 (9). Deletion plasmids were prepared by 5'-3' unidirectional deletion with exonuclease (18). Briefly, pDAV was digested with BamHI and PstI, followed by exonuclease III at 15°C (the 3' overhang of the PstI site protected the plasmid sequences from enzyme attack). Aliquots were removed from the reaction at different times, and the DNA end-filled and religated. Candidate plasmids were chosen by screening for insert sizes, and the remaining promoter regions checked by DNA sequencing.

Electroporation of trypanosomes Two different electroporation protocols were used, depending on the trypanosome life cycle stage, source of DNA and electroporation apparatus used. Method 1. Procyclic

5858 Nucleic Acids Research, Vol. 19, No. 21 trypanosomes were centrifuged at 4°C, washed once in PSGSA (9) and resuspended at a concentration of 2.107 ml-l in cold Zimmermans Post Fusion Medium (ZPFM; Bellofatto & Cross, 19). Plasmid DNA purified by alkaline lysis and CsCl density gradient centifugation (20-50 Atg in up to 50 tl Tris buffer), was added to the electroporation cuvettes (0.4 cm electrode gap) and mixed with the trypanosome suspension. Two pulses of 1.5kV at 25AtF were applied successively to each cuvette, with a BioRad gene pulser, which resulted in about 50% cell death. After 5 -10 minutes incubation on ice the contents of the cuvettes were transferred directly to culture flasks containing the appropriate medium. Method 2. (a) Procyclic trypanosomes were centrifuged, washed in ice cold phosphate-sucrose buffer (272 mM sucrose, 7 mM sodium phosphate, pH 7.2) at a density of 4.108 cells ml-1, mixed with 10 -20OAg miniprep DNA in an electroporation cuvette and placed on ice. The cuvette was then subjected to 3 double pulses of current from a Cellject apparatus (Eurogentech), consisting of an initial pulse of 400V, 74W, 40AtF, followed by a second pulse of IOOV, 74W, 2100/tF, delivered automatically with the minimum possible delay. The surviving cells (approximately 60%) were then added to 20 ml of SDM-79 and incubated at 27°C. (b) Bloodstream form trypanosomes were obtained from rats at the peak of parasitaemia by cardiac puncture, and diluted 2:1 in fresh Baltz medium (20). A buffy coat was obtained by centrifugation at 300g for 15 min and the trypanosomes removed and diluted in Baltz medium at a concentration of 4.108 cells ml-'. Electroporation was carried out using the Cellject apparatus as described above for procyclic forms except that all procedures were at room temperature. After electroporation the cells were transferred to 20 ml of Baltz medium and incubated at 370C.

Assay of CAT activity Method 1 (see above). After 18-24 hours trypanosomes were counted, centrifuged, washed with PSGSA and resuspended in 250 mM Tris (pH 8). Cells were lysed by 3 cycles of freezethaw, centrifuged, incubated at 65 °C for 5 minutes then recentrifuged and placed on ice until assayed. The CAT assay was modified from Bellofatto and Cross (19) and Clayton et al (8). Extracts from 1.107 cells were incubated at 37°C for 2 hours in a final volume of 100 A1, after adding [ring-3, 53H]-chloramphenicol (NEN Dupont), and 0.25 mM butyryl-coA (Sigma) as substrate. Labelled compounds were extracted once with lml ethyl acetate, evaporated to dryness and spotted onto silica gel plates in 20 1l of ethyl acetate. Products were separated by ascending TLC using chloroform: methanol (95:5) as solvent. TLC plates were sprayed with an autoradiographic enhancer (DuPont), and exposed for 24-72 hours at - 700C. Method 2 (see above). After 4-5 hours cells were centrifuged, washed and lysed as described in Bellofatto & Cross (19). Cell lysates were then diluted and 0.3-1.25% of the aliquots used in each assay. Reactions were carried out in a final volume of 50j1 for one hour, using (14C) chloramphenicol (Amersham) and acetyl coenzyme A (Sigma) as substrate. Reaction products were extracted using 2 volumes of xylene, evaporated to dryness, resuspended in 20d1 of ethyl acetate and subjected to TLC as described above. TLC plates were exposed for 4-24 hours without the use of enhancer. Run-on transcription Run-on transcription assays were conducted as described by Murphy et al. (21). The standard assay (1 ml) contained 500 tg

Fig. 1. The 5' environment of the T. brucei actin genes is not repeated. Southern blots of several restriction digests of genomic T. brucei AnTat 1 .3A DNA were hybridized with different probes from a cloned 13 kb SalI fragment encompassing the 5' environment of the actin genes (A, B, C, as defined in the restriction map below). Abbreviations for restriction endonucleases for all figures are: Ba=BamHI; Bg=BglII; E=EcoRI; H=HindIII; K=KpnI; S=SalI; Sp=SphI.

of DNA in isolated nuclei, 12.5 % (v/v) glycerol, 0.8 mg heparin, 5 mM spermidine, 5 mM MgCl2, 2.5 mM dithiothreitol, 10 mM Tris-HCl (pH 8), 0.5 mM ATP, UTP, CTP and 1 mCi of x-(32P)GTP (2000 Ci mmol - 1). The nuclei were usually incubated for 30 min at 30°C, conditions which give a maximal nucleotide incorporation into RNA, as defined by Coquelet et al (6).

UV irradiation UV irradiation was performed under the conditions defined by Johnson et al. (22). Briefly, the blood containing trypanosomes was diluted in Baltz medium at 37°C to reach a parasite concentration of 1 to 4.107 ml-'. Samples (125 ml) were irradiated at 254 nm (IJ sec-rm-2) in sterile square dishes (22 x22 cm) (Bio-Assay, Nunc, Roskilde, Denmark) usually for 1 min and with agitation. The irradiated cells were then transferred to culture flasks and kept in the dark, usually for 30 min, until centrifugation.

RESULTS The 5' environment of the T. brucei actin genes In T. brucei, the actin genes are present in two to four tandemlylinked copies, depending on the strain (1). Apart from a polymorphism most likely to be due to diploidy, the immediate 5' and 3' environment of the actin genes was found to be unique (1). We cloned a 13 kb Sall genomic fragment encompassing the first actin gene copy together with 11 kb of its upstream environment. Southern blot hybridization with probes derived from this clone confirmed that this region, up to about 5 kb upstream from the gene, was not repeated in the genome. After digestion by different restriction endonucleases, only a single specific DNA fragment could be detected, besides a weakly crosshybridizing band (Fig. 1, probes B,C). More than 5 kb upstream from the first actin gene copy (approximately at the level of a

Nucleic Acids Research, Vol. 19, No. 21 5859

uv/c

0

.

3

2

.

2

min. UV

2

1

0.5

31

.1 1

ER

E tgE HI(OBgB.H HB.A.S,B4 KSKB

18,

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ici

4

2

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3

4

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2 min

0 k

Fig. 2. Effect of UV irradiation on run-on transcription of the 5' environment of the T. brucei actin genes. Panel I. Run-on transcripts from nuclei of bloodstream forms, UV-irradiated for 0, 0.5, 1 and 2 min as indicated, were hybridized with different restriction digests of a 13 kb actin-specific SailI fragment cloned into the EMBL3 phage (lanes A, B and C, for HindH + KpnI, HindIll + BglII and EcoRI, respectively). The first panel shows the ethidium bromide staining of the digests, whose interpretation is given in the restriction maps below (the numbered fragments in maps A, B and C correspond to those in lanes A, B and C). The four other panels show the hybridization patterns with the run-on probes. The interpretation of the results is shown in the top restriction map, where the black boxes correspond to the Sall arms of the phage vector, the open box indicates the first copy of tandemly-linked actin genes, the thick arrows define the extent and orientation of transcripts as deduced from the data above, and the bracket shows the extent of the HindIH fragment subcloned into PUC18 for subsequent analysis. Panel II. The level of hybridization of run-on transcripts to several restriction fragments was determined by liquid scintillation counting of the relevant nitrocellulose filter areas. The ratios of labelling between probes from UV-irradiated and non-irradiated trypanosomes (UV/C) were plotted as a function of the time interval of irradiation for different DNA fragments (same numbers as in panel I) (A), or as a distance from the actin gene after 2 min-irradiation (B). In (A), the dotted line shows the level of overall run-on transcription as a function of UV irradiation, as determined by trichloroacetic acid precipitation and counting, taking 1 as a value for the non-irradiated control. In (B), the UV/C values are plotted with regard to the center of each corresponding restriction fragment. The fragments whose transcription level was analyzed are indicated in the map below. The Hindfll-BgII fragment located between -6 and -5 kb with respect to the beginning of the actin gene did not appear to be transcribed, hence no UV/C value is given.

pair of EcoRI sites: see the map in Fig. 1), the DNA was found to exist in two slightly divergent copies, as illustrated by the presence of the 11 kb HindI fragment in Fig. 1 (probe A) and by analysis of the cloned copy (data not shown). Mapping of the transcription start region by UV irradiation Moderate irradiation of trypanosomes by UV leads to both arrest of RNA elongation and inhibition of RNA degradation (6). The combination of these two effects is probably responsible for the accumulation of normally unstable transcripts which occurs at the beginning of at least some transcription units (2-6). This accumulation, easily detectable in run-on transcription assays, can thus be taken as a marker for the proximity of a promoter. Run-on transcripts from non-irradiated and UV-irradiated bloodstream forms were hybridized with restriction digests of the cloned genomic SalI fragment which extends to 11 kb upstream from the first actin gene. The data, shown in Fig. 2 (panel I: hybridization patterns; panel II: graphic plots of UV/control ratios), indicate that UV irradiation induced a twofold accumulation of transcripts from a region located about 4 kb upstream from the actin genes (fragment 5 in lane A, 6 and 7 in lane B). That this region may contain the transcription start site was further supported by the observation, illustrated in Fig. 2, that the region with the apparent stimulation of transcription by UV is immediately preceded by a seemingly silent stretch of DNA (fragment 8 in lanes A and B), itself located downstream from a region whose transcription is severely inhibited by UV (fragment 2 in lane A, 3 in lane B, 3 and 4 in lane C). A

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Fig. 3. Steady-state transcripts from the presumptive actin gene promoter region. Northern blots of 5 yg of poly(A)+ RNA from either non-irradiated or 1 min UV-irradiated procyclic and bloodstream forms (p, pu, b, bu, respectively) were hybridized with the A, B and C probes defined in Fig. 1, and with the actin gene probe as a control. The autoradiograms in the first three panels were exposed 5-fold longer than the fourh.

straightforward interpretation of the data is that the region analyzed in Fig. 2 contains the end of an uncharacterized transcription unit, followed by the beginning of the actin gene unit, both being oriented in the same direction, as shown schematically by the thick arrows under the restriction map of Fig. 2 (panel I).

5860 Nucleic Acids Research, Vol. 19, No. 21 Bomt Hi

61 1

.0

181 241 301 3 61

4221 481 541 601 661 7 21 781

841 901 961

1C2' 1081 114;

1201

Fig. 4. Transient CAT activity assays driven by the actin promoter. Bloodstream and procyclic forms were electroporated with different plasmid constructs, and the level of CAT activity was determined after 4-5 h. The photograph shows the results of a typical experiment using method 2 (see Materials and Methods). The plasmids are pDAV (lane 1), progressive deletions from pDAV (pDAVdI to pDAVd8, in lanes 2 to 9, respectively) and pDAVr which contains the promoter region in inverted orientation (lane 10). The schematic to the left illustrates the relative length of each deletion. The numbers to the left represent the size of the remaining fragments, in base pairs, with respect to the Hindul site at the 3' end of the cloned actin promoter fragment (vertical bar). The numbers to the right represent the levels of CAT activity of each construct compared with pDAV (activity = 100%), in procyclic (P) and bloodstream (B) forms.

These run-on data were confirmed by Northern blot hybridization, which showed that the region immediately preceding the putative actin gene transcription start does not appear to be transcribed into stable RNA (Fig. 3, probe A), while the whole region downstream, to the actin genes, is transcribed (Fig. 3, probes B, C, and ref. 1). Also in accordance with the run-on data was the observation that the amount of steady-state RNA specific to the region immediately downstream from the untranscribed stretch is increased following UV treatment (Fig. 3, probes B, C), while this is not seen for the actin gene region, which is more distant from the transcription gap (Fig. 3, actin probe, and ref. 1). Transient activity assays driven by the actin promoter A 1.2 kb BamHI-HindIII fragment from the putative actin gene promoter region was cloned upstream from the bacterial gene for chloramphenicol acetyl transferase (CAT), by replacement of the VSG gene promoter in plasmid pD5V (9). This plasmid construct (pDAV) was electroporated into both bloodstream and procyclic trypanosomes. Cell extracts from both forms showed similar levels of CAT activity (Fig. 4, lane 1). No activity was detected when the fragment was inserted in a reverse orientation (Fig. 4, lane 10). The promoter sequence was subsequently deleted by controlled exonucleolytic digestion. The removal of the first 180 bp induced an apparent increase in activity (lane 2). Further deletion led to the progressive disappearance of CAT activity, the reduction starting around -740 bp with respect to the 3' end of the inserted fragment (Fig. 4, lanes 3-9).

Nucleotide sequence of the actin gene promoter Fig. 5 shows the nucleotide sequence of the 1.2 kb BamHIHindlIl fragment encompassing the actin gene promoter. This

GGATCCCAAA ATGGCGTCCC CTCCGATGC2fl GGGCGGCCAT GGCTGGCCGC CTTCGCAACA GTGTGGGGAT TGGCCTGTAG TTATGACCT CTTACTTTTG

CACTGCTITCG GT TOTOT1 T GGCCTGTTGT CATCTCCGCT CTCAGGTGTG

TCAAAGGTT1' ,42 TTCCA2CTAAC GAGCGTTTam

TGGGTGTCGA AATCTGGTGG

GCATGATAAG GTTGCTTCGG TCTTTGAAGG ATCAATCGTG AAAGAAGTTG

3TTTGTCTTA

ACCTCCTI-I

CGTAGGCGGC d4

GAATGCG:GAT

CCGTCGCCAG GGGGCTGGGT GGTGTGC^A--GGC^mC CCGGCTGCTC TGATGCTCvG GGCC-GGGGT GCATGvGvGG ATTAAAGGTT CATACGCCAG TGACGGTGAT GACTCCACGT CTGCTTTGGC ACCGCGGCTG GGGCCCGCTG TGCGCAGATG CGAGAAACCG GAGGCTCGCG TGGGTCCGAT GCCCGTGGAG GGCCGTGCTG GTGTTGGTGC TAGTGGTGTT GTGCOGGACC CTCATGTGCG GACCGGCTCG TCGTGAGTGG TGGGCGGACG GGGATGCTGG -TGTATCTGT TGCCGCGACG CGGCCXCACC CTTATTTTCG TGTTTTGT TTGT TTTAC TGGGGTTCGG CATAGTTACG GTGTCGGTC- TTCGGCGCCT GTCGGCGG= GCGCTGGTGC AGGTCGC=TT GAACGGGAGG GCCACCACA CTbCGGTGTG TGTGTGTGGC CCCCCAC`TTC GCCGCTGGTC GTAT'IGTCG GGCCGGGGGC ACTCGGGGGG CGTGTGACGT CGAGGATGAG CGCCCCCCTT TITTTTAAAG GGGGGGGCCG CCACCGTTGG TGTGGCqCCT =TGTTGGGTG ATGGGTGACO GTATTTGGTT GGTTCTGTCG CT,TGGAGGCT ATGCTGAAAC CATATATGTG TGCGTGCGCC TGCATGACCT AGGCGATGCO

GAGAGGGAGG =TTGTACCGT TTGTTGGTTG CCT-TGTGCTG CCGGTGCCTA GAAGCACGCA TTTTCTTAGA AGTATGTGCC CC=. TCrC.CC CGTOGGACO- TT=CA=ACO CGTTTGTGTT GAAAGCAAAA AATTCCCCTT CGCCTTCA C TCTCTGTO AGGATATCGG CAGGATGGCG TTGTTCGT GCT=CGTTG TGG7TACATG TACGAA=.TG CGGTATCGAA CAGCTATTGT AGGAAGCTT14 bodS8 Mind 911

Fig. 5. Nucleotide sequence of the 1. 2 kb BamHI-Hindl fragment encompassing the actin promoter. Underlined stretches 1 and 2 are large direct repeats. The opposite arrows centered around position 820 indicate a palindromic structure. Positions dl to d8 mark the 5' limits of successive deletions, corresponding to positions: -917, -722, -621, -540, -415, -352, -220 and + 137, with respect to the putative transcription start site (asterisk), which was determined by primer extension (Fig. 6). The wavy line defines the extent of the region complementary to the oligonucleotide used for primer extension (Fig. 6).

sequence does not show significant homology to other known trypanosome promoters. It contains two pairs of large direct repeats (10 bp and 9 bp, respectively underlined 1 and 2), as well as a pair of inverted repeats (opposite arrows) with a high potential of forming a stable hairpin structure containing a 13 bp G/C-rich stem and a loop of five thymidines (base pairing energy: -28 Kcal/mol in IM Na+ (pH 7.0) at 25°C).

Mapping of the transcription start A primer extension experiment was conducted by reverse transcription of RNA from either UV-treated or untreated cells, after annealing with an oligonucleotide from the 3' terminus of the 1.2 kb BamHI-HindIII fragment (complementary to the sequence underlined with a wavy line in Fig. 5). The results, shown in Fig. 6, indicate that a major transcription start is present 124 bp upstream from HindI, in both bloodstream and procyclic forms. The extra bands that occur in the procyclic and AnTatl.3A lanes appear to be strain specific as the procyclics were derived from the EATRO 1125 stock, as was the AnTatl .3A bloodstream clone. These bands are not present in the T b. gambiense AnTatl 1.17 bloodstream variant.

DISCUSSION This paper reports the characterization of the first transcription promoter for a constitutively-expressed housekeeping gene of T. brucei. The promoter region was localized by trypanosome irradiation with UV, which leads to both blocking of RNA elongation and a reduction in the rate of RNA degradation (6,23). The combination of these two effects results in the accumulation of promoter-proximal transcripts in at least some transcription units (2-6,8). This UV-induced accumulation was thus taken as an indication of the approximate position of the actin promoter.

Nucleic Acids Research, Vol. 19, No. 21 5861 c

11.17

Pr

3ATCGA

-80

-70

-60

-50

-40

-30

-20

-10

+1

ACT: CGCGA TGCTGAACCA C0CATTCT TACAAGTATC TGCCC=r_CT CCCTCTC CACCTCA CTTGCGTTTG TGTTGAAAGC AAAA

VSG:

AACCT TCTAAAAGAA TCATATCCCTMAff4CACAC

CATTATAICL2&QVGGAG GTTAT&TLG AAATCTCGGA TATCAGACTC

ACCC

Fig. 7. Comparison of the region immediately upstream from the presumptive transcription start (+ 1) in the actin (ACT) and VSG promoters. Direct repeats are underlined.

UV

A* A7 fi A~ A-

A ~ .A"

;

Fig. 6. Mapping of the start of the actin gene transcription umit. Primer extension wass conducted on 5 /tg of poly(A) -(A ) or poly(A) + (A t) RNA from procycic (Pr) or bloodstream forms (clone AnTat 11.17 from T b. gambiense ( 1. 17), or clone AnTat I.3A from T' brucei (3 A)), after annealing with 150 ng of a (2) labelled 30-mer oligonucleotide from the 3' end of the 1.2 kb BamHI-HindllI fragment (see Fig. 5). When indicated, the RNA was from trypanosomes UVirradiated for 1 min (uv). As a control, 5 usg of yeast tRNA was processed in the same way (C). A sequencing reaction performed on plasnmid pDAV from the same primer was run on the same gel to map the beginning of the largest discrete cDNA.

In an 11I kb sequence upstream from the actin genes, only one region exhibited the expected transcription pattern. That this region contained a transcription start was further suggested by the presence, immediately upstream, of an apparently untranscribed stretch of DNA. Moreover, the latter was preceeded by what appeared to be the end of another ftrascription unit, as judged by its strong level of inhibition by UV. Further strong evidence that the region defined by UV mapping encompasses a transcription promoter was provided by its ability to direct expression of the CAT gene in plasmid constructs electroporated into both bloodstream and procyclic forms of the parasite. This analysis confirms that the dual effect of UV on run-on transcription can usefully help to define the extent of transcription units in T. brucei. While the level of accumulation of promoter-proximal RNA was found to be about 5-fold in the rDNA, VSG and procyclin gene transcription units (6), it was only 2-fold in the actin unit. This could be tentatively correlated with two features which distinguish the latter unit from the three others: as opposed to the rDNA, VSG and procyclin genes, the actin genes are

transcribed by a polymerase sensitive to a-amanitin, and their rate of transcription, as determined by run-on assays, is reproducibly 5 to 10-fold lower. This last characteristic seems to be shared by other transcription units transcribed by an aamanitin sensitive RNA polymerase, such as those of TBA1, a P-ATPase gene of T. brucei (24), and several other unidentified trypanosome genes (E.P., unpublished observations). One possible interpretation of the data is that the density and / or activity of the a-amanitin sensitive RNA polymerase is 5 to 10-fold lower than that of its c-amanitin insensitive counterpart(s). Hence, the rate of promoter-proximal RNA enrichment following UV would be correspondingly lower. Alternatively, the turnover rate of promoter-proximal transcripts may be lower in the actin gene transcription unit than in those of the rDNA, VSG and procyclin genes. The actin gene promoter is located about 4 kb upstream from the first actin gene open reading frame. As the region between the promoter and the actin genes is transcribed into stable polyadenylated RNAs, it may be hypothesized that it contains additional genes, and that the actin gene transcription unit is polycistronic as has been observed for many other trypanosome genes (7). This hypothesis is further supported by the observation that a major polyadenylated transcript seems to be processed from the 3' extension of a large actin mRNA precursor (1). The actin genes may thus be flanked by other coordinately transcribed genes. As yet however, we have no indication of the nature or exact location of these putative genes. From deletion analysis, the cloned region from the actin gene promoter seems to comprise three main domains: the removal of the region between -1100 and -900 with respect to the transcription start leads to stimulation of CAT expression; progressive deletion of the region between -900 and -600 does not significantly affect the level of activity; while deletion starting from -600 progressively reduces CAT expression. Further experiments are required in order to determine if these effects depend on the presence of enhancers/silencers. A similar pattern was seen in both life cycle stages, although maximal stimulation in bloodstream forms was threefold, as opposed to fourfold in procyclic forms (Fig. 4). We do not know if the levels of activity obtained in the two life cycle forms reflect the true level of actin gene promoter activity in vivo, as we have no way of comparing the efficiency of electroporation in the two forms. Furthermore, it has been shown that different gene flanking sequences can affect the levels of CAT expression in trypanosomes (9, 12). Therefore, direct comparisons of promoter activity in bloodstream and procyclic forms are not possible. The nucleotide sequence of the actin gene promoter does not share any obvious homology with transcription promoters from other eukaryotes. In addition, no significant homology could be found with the other trypanosome promoters characterized so far, which all direct transcription by a-amanitin insensitive RNA polymerase(s). However, if the actin and VSG gene promoter sequences are aligned beginning with their respective transcription starts, three direct repeats of 5 to 8 nucleotides are present at

5862 Nucleic Acids Research, Vol. 19, No. 21 approximatively the same locations in both cases, although their sequence differs (Fig. 7). This observation may be significant, as in the case of the VSG promoter we could demonstrate that the first repeat is crucial for promoter activity (A. Pays, D. Jefferies and E. Pays, unpublished data). In the case of the procyclin promoter also, short repeats have been noted (10), but their relative position with respect to the transcription start may not be the same as illustrated here (5).

ACKNOWLEDGEMENTS This work was supported by the Belgian 'Fonds de la Recherche Scientifique Medicale' and research contracts with the 'Communaute Francaise de Belgique' (ARC 89/94-134), the EEC (TDS-M-023B) and WHO. It was also funded by the Agreement for Collaborative Research between ILRAD (Nairobi) and Belgian Research Centres. D.J. is the holder of a Wellcome Trust Training Fellowship in Tropical Medicine.

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