Molecular and Biochemical Parasitology, 43 (1990) 133-142

Elsevier

MOLBIO 01409

133

A cyclic AMP inducible gene expressed during the development of infective stages of Trypanosoma cruzi

Stephen Heath, Sara Hieny and Alan Sher

Immunology and Cell Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A.

(Received 26 February 1990; accepted 11 June 1990)

By using a subtractive hybridization strategy, we have identified a gene (TC26) that is expressed in metacyclic and tissue culture derived trypomastigotes of Trypanosoma cruzi but not log stage epimastigotes and is induced during the differentiation of metacyclic stages in vitro. In contrast, the TC26 transcript is absent from stationary phase epimastigotes of a strain that fails to undergo metacyclogenesis under the same culture conditions. Transcription of TC26 can be induced in epimastigotes by incubation with cyclic AMP and cyclic AMP analogues but it is inhibited by activators of cAMP dependent phosphodiesterases. Cyclic AMP fails to enhance tubulin gene expression in the same parasites. While present in the genome in multiple copies, the TC26 gene is expressed as a single mRNA species of approximately 5 kb. Computer analysis of the sequence of a 650-bp cDNA clone revealed no significant homologies at either the nucleotide or amino acid levels with other known proteins. Possible roles for the TC26 gene product in metacyclogenesis are discussed.

Key words: Trypanosoma cruzi; Metacyclogenesis; Differentiation; Cyclic AMP; Transcription

Introduction

Trypanosoma cruzi undergoes a complex series of morphogenetic changes throughout its life cy- cle [1,2]. A major developmental transition oc- curs when the noninfective, replicating, epimasti- gote (EPI) stage transforms within the hindgut of the triatomid insect vector to an infective, non- dividing metacyclic trypomastigote (MT) which is then eliminated in the insect feces. Metacyclogen- esis is of crucial importance as it preadapts the parasite for survival within the vertebrate host, allowing it to evade major defenses such as the

Correspondence (present) address: Stephen Heath, Dept. of Biological Sciences, University of Salford, Salford M5 4WT, England, U.K.

Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank TM database with the accession number M34062.

Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; LIT, liver infusion tryptone; BESM cells, bovine embryo skeletal muscle cells; EPI, epimastigote; MT, metacyclic try- pomastigote.

alternative complement pathway [3] and the res- piratory burst of phagocytes [4]. Upon culture to stationary phase, EPI of many T. cruzi strains dif- ferentiate into metacyclic forms that are indistin- guishable in their biological behavior from insect- derived MT [1,2,5].

Numerous studies have attempted to analyze the process of metacyclogenesis in T. cruzi. Of particular interest are recent data implicating the ubiquitous intracellular second messenger cyclic AMP (cAMP) in the control of MT differentia- tion in vitro. Thus cAMP levels are elevated in MT with respect to EPI [6] and addition of ex- ogenous cAMP or cAMP analogues accelerates metacyclogenesis in vitro [7].

The aim of the present study was to define the genetic changes occurring during metacyclogene- sis in order to further elucidate the mechanism(s) controlling differentiation. Towards this end, T. cruzi genes expressed preferentially at the meta- cyclic stage of development were identified by a process of cDNA cloning and subtractive hy- bridization. In this report, we describe the charac-

0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)

I34

terizalion of one developmentally regulated meta- cyclic gene, which is of interest because its ex- pression can be induced in EPI by cAMP and cAMP analogues.

Materials and Methods

Parasites. The Sylvio X10/4 clone and Tulahuen strain of T. cruzi were kindly provided by J. Dvo- rak (National Institutes of Health, Bethesda, MD). Epimastigotes were maintained by serial passage in LIT medium (Oxoid, U.K.) containing 10% (v/v) fetal calf serum, 0.02 mg ml - I hemin, 100 U ml -~ penicillin and 100 lzg ml - t streptomycin at 26°C. When log phase EPI were required, para- sites were harvested at a culture density of less than 1 × 107 m1-1.

In vitro derived metacyclic trypomastigotes (CMT) were produced in stationary cultures as previously described [8]. CMT were partially pu- rified from EPI by DEAE cellulose chromatogra- phy [9]. Lysis of the remaining EPI was accom- plished by treatment for 45 min at 37°C with 10% (v/v) guinea pig complement in DMEM at a den- sity of 1 x 105 parasites m1-1. The resulting CMT populations contained less than 1% EPI.

Nucleic acid extraction. Total RNA from T. cruzi was extracted using the guanidinium thio- cyanate/CsCl procedure [10]. To ensure that RNA preparations were not contaminated with small quantities of DNA, a final digestion with RNase- free DNase (5'prime 3'prime, USA) was per- formed. Poly(A) + RNA was purified from total RNA using poly-U Sephadex (BRL, U.S.A.) ac- cording to the manufacturer's instructions.

cDNA cloning and isolation of metacyclic eDNA clones. X10/4 CMT cDNA was synthesized from a poly(A) + RNA template by random hexamer priming using MLV reverse transcriptase (BRL, USA) as described previously [11,12] and inserted into the bacteriophage vector Agtl 1 using standard procedures.

Approximately 105 recombinant clones from the CMT cDNA library were screened in duplicate with a single stranded [32P]cDNA probe highly enriched in sequences coding for CMT specific proteins. The [32p]cDNA probe was constructed

as described by Sargent and Dawid [13]. Briefly, 32P-labeled single-stranded CMT cDNA was hy- bridized exhaustively to a large excess of EPI RNA (Rot ~ 2000 mol l - I s-~). The hybridiza- tion mixture was then applied to hydroxylapatite at 60°C and 32P-labeled single-stranded cDNA was eluted with 0.12 M NaHzPO4, pH 6.8. The CMT specificity of each probe preparation was checked by RNA dot blot analysis. Hybridization and hydroxylapatite purification procedures were repeated three times to ensure an optimal signal to background ratio.

When screening the metacyclic cDNA library [14], hybridizations were performed at 42°C in 5 x SSC (20 x SSC= 3 M NaC1/0.3 M Na-citrate, pH 8.3)/50% formamide and high- stringency washes were carried out at 68°C in 0.1 x SSC/0.1% SDS as described previously [15].

Hybridization analyses. DNA probes for hy- bridization analyses were obtained by restriction digestion and gel purification of recombinant in- sert DNA and labeled to high specific activity (>109 cpm ll, g - i) using a random priming label- ing technique [16].

For Northern analysis RNA was electrophoresed in 1% agarose gels containing 2.2 M formalde- hyde and then transferred to Genescreen Plus (NEN) according to the manufacturer's instruc- tions. Filters were hybridized with probe ATC26 in 6 x SSC/0.1% SDS/50% formamide at 42°C and stringent post-hybridization washes were per- formed at 68°C in 0.1 x SSC/0.1% SDS. As an independent assessment of loading RNA con- centration, a duplicate gel was run and stained with ethidium bromide to visualize the ribosomal bands.

cAMP-induced transcription. Epimastigotes were grown at 26°C in LIT medium to log phase. The parasites were washed by centrifugation at 1500 x gay and resuspended in fresh LIT medium con- taining a range of concentrations (0-100 ltM) of either cAMP, dibutyryl cAMP, 8-bromo-cAMP, indomethacin, dibutyryl cAMP + 50 p.g ml-~ im- idazole. Parasites were cultured for a further 48 h at which time an aliquot of culture, containing 105 parasites, was removed and cytoplasmic RNA extracted by lysis of parasites in lysis buffer (1%

NP-40/200mM NaC1/50 mM Tris-HC1 pH 7.4/1 mM EDTA) and centrifugation at 10000 x gay to remove the nuclei [17]. The aqueous phase was extracted once with phenol/chloroform and once with chloroform. Formaldehyde and NaCI were added to 2.2 M and 1 M, respectively, and the RNA preparations were then transferred to Gene- screen plus nylon filters. Blots were hybridized for Northern analysis, as described previously.

DNA sequencing and analysis. Positive A phage clones were rescreened and replated to homo- geneity and inserts were isolated by digesting recombinant construct DNA with an appropri- ate restriction enzyme. Inserts were gel purified and subcloned into EcoRI-digested, dephospho- rylated pUC13 (Pharmacia). Double-stranded se- quencing was performed directly in the collapsed plasmid using [ot-35S]thio-dAZP and Sequenase (US Biochemicals USA). To elucidate internal se- quences distal to the cloning sites we generated a series of subclones with overlapping restric- tion fragments, in combination with short (17- mers) synthetic oligonucleotide primers (Synthe- cell, USA). The resulting sequences were ana- lyzed using PC/GENE (Intelligenetics) software. All homology searches against the NBRF pro- tein database and GenBank were performed on a DEC 10 mainframe computer using software pro- vided by the National Institutes of Health (SEQF and SEQFP).

Results

Identification and characterization of a metacyclic specific cDNA clone. Approximately 105 recom- binant Agtl 1 clones from a CMT cDNA library were screened with a metacyclic-specific subtrac- tive cDNA probe. From this initial screen we identified nine positives with insert sizes ranging from about 400 to 900 bp. Three of the original nine clones were found to be identical and one (ATC26) was chosen for further characterization because it gave the strongest hybridization sig- nal difference with the original subtractive cDNA probe when compared to EPI cDNA clones. The specificity of TC26 is shown in Fig. 1. This slot blot was performed on cytoplasmic RNA on a cell-equivalent basis in order to eliminate any ar-

EPI CMT

135

. m l O 8

10 7

~ 1 0 6

10 5

~ 1 0 4

~ 1 0 3

Fig. 1. Hybridization of TC26 to cytoplasmic RNA from T. cruzi CMT and EPI at different cell densities. Hybridization and washing conditions were as described in Materials and

Methods.

tifacts resulting from stage differences in absolute levels of extractable RNA. The data clearly show that TC26 transcripts are more abundant in CMTs. The signals observed with EPIs at densities of 107 and 108 probably reflect the presence of contami- nating CMTs (usually ranging from 1-2%) in the EPI preparation.

Hybridization of the ATC26 insert to Northern blots of CMT and EPI RNA, demonstrated that the gene is transcribed in CMT to yield a single mRNA species of approximately 5 kb (Fig. 2). No hybridization of the probe was observed to EPI RNA in this experiment.

Developmental regulation of TC26. In order to study transcription of the TC26 gene during meta- cyclogenesis in T. cruzi, we allowed log stage EPI to differentiate in Grace's medium for a period of

136

a kb

9.5__ /---

7.5

4.4__

EPI CMT

Fig. 2. Northem analysis of TC26. mRNA was isolated from both EPI and MT and 1 /tg of poly U selected RNA per lane was electropboresed in a 1% agarose formaldehyde denaturing gel and transferred to nylon filters. Hybridization with 32p_ labeled ATC26 and washing conditions were as described in

Materials and Methods.

8 days (Fig. 3A). During this period cytoplasmic RNA was isolated from aliquots of the culture at 48 h intervals and immobilized on nylon mem- branes. Hybridization of [32p] ATC26 with RNA isolated from differentiating EPIs, confirmed that the TC26 gene is not transcriptionally active in noninfective log stage EPI, but is expressed af- ter 48 h and thereafter as differentiation to CMT proceeds during growth to stationary phase (Fig. 3B). This induction is not a function of the growth cycle per se since stationary phase cultures of Tu- lahuen strain T. cruzi, which fail to differentiate to CMT under these conditions, also fail to express the TC26 transcript (Fig. 3C). Nevertheless, the transcript is present in Tulahuen strain tissue cul- ture trypomastigotes (TCT), derived by infection of BESM cells, confirming the presence of the gene in parasites of this strain and demonstrating its expression during alternative conditions of try- panosome differentiation. Thus, these data clearly demonstrate that transcription of the TC26 gene is directly associated with the biological differen- tiation of EPI to CMT.

cAMP-induced transcription. To evaluate the ef- fects of increased levels of extracellular cAMP on the expression of TC26 transcripts, EPI were

grown to early log phase at 26°C and then incu- bated with various cyclic nucleotides for 48 h. Cy- toplasmic RNA was then extracted and hybridized with ATC26. Fig. 4 shows that the metacyclic specific g~ne ATC26 can be induced in nonin- fective epimastigotes by addition of either dibu- tyryl cAMP, 8-bromo-cAMP and to a lesser ex- tent, native cAMP. Furthermore, an increase in the hybridization signal density was shown to oc- cur in response to increasing cAMP concentration suggesting that this effect is dose dependent. In-

A 80" 100

~ o 80 • x 60

~ , 60 .=

-*" 4o

20 0

0 2 4 6 8 10

days in Graee's cul ture

B 0 2 I I

II

I I I

X1014

Tulahuen Log

S t a t

TCT ~

Fig. 3. Stage specificity of TC26. (A) In vitro differentiation of T. cruzi clone X10. Early log-stage EPI grown in LIT medium were transferred to Grace's Insect Medium and cultured at 28°C for 8 days. At 48-h intervals, total numbers of para- sites and relative percentages of metacyclic trypomastigotes were assessed by microscopic examination. (B) Transcription of TC26 gene during metacyclogenesis of XI0 strain MT. At 48 h intervals the extent of TC26 transcription was assessed as follows: cytoplasmic RNA was extracted and transferred to nylon membranes. The RNA blots were then hybridized with 32p-labeled TC26 cDNA probe at 42°C in 50% for- mamide and final high-stringency washes performed. (C) Ab- sence of TC26 transcript in stationary phase Grace's cultures of T. cruzi Tulahuen strain. Cytoplasmic RNA was extracted from log and stationary phase parasites, hybridized to ATC26 and washed as described above. Tissue culture derived trypo- mastigotes (TCT), which were used as a positive control, were

obtained by culture in BESM cells at 37°C.

(uM) A C D F

137

100

10

0

Fig. 4. cAMP induction of TC26 transcription. Log stage EPI (x l0) were incubated in LIT medium for 48 h in the presence of decreasing concentrations of (A) cAMP; (B) dibutyryl cAMP; (C) cAMP + 50 #M Imidazole; (D) 8-bromo-cAMP; and (E) indomethacin. As a control for these experiments (F); the relative levels of tubulin transcripts from dibutyryl cAMP-stimulated EPI (B) was assessed using a 32p-labeled c~ and B tubulin probe. Cytoplasmic RNA extraction, hybridization with ATC26 and

washing conditions were as described in Materials and Methods.

domethacin, which is thought to exert its effects indirectly by inhibiting prostaglandin synthesis causing a concomitant increase in the intracellular levels of cAMP [18], was also shown to induce transcription of TC26 (Fig. 4, lane E). Moreover, addition of an excess of the phosphodiesterase ac- tivator imidazole, which converts cAMP to 5 I- AMP, inhibited the induction of TC26 by dibu- tyryl cAMP in EPI (Fig. 4, lane F). Nevertheless, the effect of cAMP on TC26 does not appear to be the result of a generalized effect on transcrip- tion since tubulin gene expression in EPI was not enhanced or diminished by dibutyryl cAMP treat- ment (Fig. 4, lane F).

Sequence analysis. Using the ATC26 clone as a probe, the metacyclic cDNA library was re- screened in order to identify identical clones con- taining larger inserts, cDNA clones isolated from the Agtll library were subcloned into pUC13 and sequenced directly. Sequence analysis of the largest cDNA clone (approx. 650 bp) identified a single major open reading frame of approximately 213 amino acids (Fig. 5). A search of GenBank and the NBRF protein databases (October, 1989) failed to reveal any significant homologies with TC26. Computer analysis of the amino acid se- quence using PROSITE [19] identified three pu- tative phosphorylation sites and two putative gly- cosylation sites.

Discussion

Metacyclogenesis of T. cruzi in stationary cul- ture provides an excellent model for studying the genetic control of parasite differentiation [5,8]. In this system, the process of vertebrate adapta- tion can be studied in vitro and large numbers of both infective and noninfective forms generated for molecular analysis. In the present study we have used a subtractive hybridization approach to identify a gene expressed during the development of metacyclic stages. This gene is of interest be- cause its transcription is regulated by cAMP, an agent previously shown to influence the process of metacyclogenesis itself.

Previous studies on developmentally regulated molecules of T. cruzi have focused on the iden- tification of stage-specific antigens expressed in trypomastigotes but not epimastigotes. Thus, try- pomastigote specific antigens of 75 [20], 160 [21 ], 85 [22] and 90 kDa [23], have been identified in various T. cruzi strains and the genes for two of these molecules (the 85- and 90-kDa species) have been cloned and sequenced. More recently, an- other 85-kDa trypomastigote specific surface anti- gen gene has been identified which is distinct from the previously identified gene product of the same molecular size [24]. The most frequently used ap- proach for identification of trypomastigote surface antigens has been by antibody screening of cDNA libraries prepared from mRNA isolated from this parasite stage. In contrast, no selection for anti- gens or expressed proteins was inherent in the

138

5' 100 nt 3'

~ , -

I I I I I I I I TC26

i0 20 30 40 50 60

I I I I I I * ACCCCACCAGACCAACTGGTAATGGTAGCGACCGGCGCTCAGCTTGGAATTCCGTGCGCTGCTTCTAGC T P P D Q L V M V A T G A Q L G I P C A A S S

70 80 90 I00 ii0 120 130

I I I I I I I CCTAGAGAAGCTGATGATCCCCCGTATCCGACACAGGCGTACAACCCTGCTTGTGGTTACGGACAGTCA P R E A D D P P Y P T Q A Y N P A C G Y G Q S

140 150 160 170 180 190 200

I I I I * I I I GTCTCTTCTAGCGGCTCTAAACAAGGGCCCGCTCAATCAGACAGACTGGACGGAGGATCAGATCTGGCG V S S S G S K Q G P A Q S D R L D G G S D L A

210 220 230 240 250 260 270

I I E I I I I GCGTCTCTTGACACTGACGTGTGCTGGCTGGTCGTGCACCTGCAGTTTTGTTACGGACATTGTGGAGTA A S L D T D V C W L V V H L Q F C Y G H C G V

280 290 300 310 320 330 340

I I I I I I I # CATGCTAACGAGCTTGCAGATCAGTATGCGAGGAACTATGGAAAGTGGACAATACACGGAGCAAGGAAT H A N E L A D Q Y A R N Y G K W T I H G A R N

350 360 370 380 390 400 410

L I 1 I I I I CGCACCTTTATGGCATACGGATCTGCTGACGTGTTTTACTACCCAGCTCACCAACAAGTGGCGTACTAC R T F M A Y G S A D V F Y Y P A H Q Q V A Y Y

420 430 440 450 460 470 480

*I I I I I I I CATTCGTCAAGACACTCATCGCTACCTGCTTTGCGGCACAAGGCCATCAGATCTCGCGGTAAGGACCTG H S S R H S S L P A L R H K A I R S R G K D L

490 500 510 520 530 540 550

I L I I I I # I ATCACTCAGGAAGTTCTACACCGTCAGAACTGGTTCACCTCGCAAGGGCAAGGTGCGGGGAATCTGAGC I T Q E V L H R Q N W F T S Q G Q G A G N L S

560 570 580 590 600 610 620

I I I I I I I TCTGGGGCCGACTATCTGGGCCGTGAGAGATTGCACGAACCAATGCCGTTTCTGCAACATCTCACCGGA S G A D Y L G R E R L H E P M P F L Q H L T G

630 I

ACAGTCTGCATATATAT T V C I Y

Fig. 5. (A) Nucleotide sequence and restriction map of ATC26 cDNA clone. Immediately below the nucleotide sequence is the deduced amino acid sequence showing putative cAMP-dependent protein kinase phosphorylation sites (*) and glycosylation sites (#).

subtractive hybridization approach employed in the present study. Instead, any gene preferentially transcribed in CMT could be conceivably selected by this procedure. Given the similarity in the in vitro translation profiles of CMT and EPI [25], the number of CMT specific transcripts is likely to be relatively few.

The CMT specific TC26 gene we have char- acterized is expressed as a 5-kb transcript. The sequence of the cDNA clone reveals a single major open reading frame of 213 amino acids (Fig. 5). However, neither the nucleotide or trans- lated protein sequence have significant homol- ogy to sequences in the data bank. Moreover, the ATC26 clone failed to express a detectable recom- binant protein (data not shown), and, therefore, the identity of the TC26 protein for the moment re- mains undefined. Genomic sequencing and further cDNA expression studies are in progress in an at- tempt to formally identify the TC26 gene product.

The TC26 gene is expressed in purified CMT as well as tissue culture-derived trypomastigotes (TCT) but not in log-stage EPI. While its tran- scription is initiated during growth to stationary phase, stationary phase Tulahuen cultures, which fail to undergo differentiation to CMT, do not express the gene, a finding which argues against the induction of TC26 being linked solely to the cell cycle. More formal proof of the gene's stage specificity was not possible because of the lack of a suitable procedure for separating stationary phase EPI from CMT and assaying TC26 tran- scription in the former stage.

A major goal of studies on metacyclogenesis has been the elucidation of the signals which trig- ger parasite differentiation. Previous results have argued that factors such as pH [26], amino acid levels [27] and the accumulation of metabolites [28] can play a role in stimulating transforma- tion of EPI to MT. Pertinent to our findings is the recent evidence for a role of cAMP in the induction of metacyclogenesis. In these studies, cAMP and cAMP analogues were shown to ac- celerate the generation of CMT in culture [7]. In addition, CMT have been found to contain higher intracellular levels of cAMP than EPI [6]. Since cAMP has been detected in the urine and malphigi secretion fluids from Rhodnius prolixus, an inver- tebrate vector of T. cruzi [29], it is possible that

139

this molecule could play a role in the induction of metacyclogenesis in vivo.

As the findings reported here indicate, transcrip- tion of the TC26 gene can be induced in EPI by high concentrations of cAMP and lower levels of cAMP analogues. The greater effect of the ana- logues (e.g., dibutyryl cAMP) may relate to their enhanced capacity to be transported across the parasite membrane or to their enhanced affinity for surface cAMP receptors. The effect of cAMP on TC26 transcription appears to be specific since no increase in tubulin gene expression by EPI was observed under the same conditions of cAMP in- cubation. Thus, TC26 may belong to a subset of cAMP inducible genes expressed during differen- tiation of EPI to MT.

cAMP regulates RNA levels of several eu- karyotic genes such as somatostatin [30], pre- proenkephalin [31 ], prolactin [32] and vasoactive intestinal peptide [33]. Like TC26, all of these genes are positively regulated at the level of RNA accumulation; however, other genes have been described which are under negative cAMP con- trol [34]. Numerous hypothetical mechanisms for induction of transcription in eukaryotes by ex- tracellular cAMP have been proposed. Most of them suggest a cascade of events which involves a cAMP binding protein, a cAMP-dependent pro- tein kinase and a phosphorylated nuclear factor [35,36]. Some of these components have been de- fined in T. cruzi including a surface-associated cAMP binding protein [37] and an adenylate cy- clase activity [7]. While it is probable that the induction of TC26 gene function involves this type of cascade, other levels of control (e.g., post-transcriptional stabilization/destabilization of mRNA) have not been ruled out. In addition, in- creasing evidence points to the possibility that changes in the 3' region of mRNA can drastically alter the half-life of mRNA although the rate of transcription remains constant [38]. A major clue to the functional control of TC26 gene expression would be provided by the detection of a consensus cAMP responsive element in the promotor region of TC26. The search for this domain is a major goal of our current genomic sequencing studies.

While cyclic AMP has previously been impli- cated in the control of differentiation in trypanoso- matids [39-41], the ATC26 clone characterized in

140

the present study represents to the best of our knowledge the first cAMP regulated gene iden- tified in these unicellular organisms. It hopefully will serve as a useful model for examining both signal transduction and transcriptional control in parasitic protozoa of this group.

Acknowledgements

We would like to thank Dr. Thomas McCutchan for his advice and criticism, Dr. Juan C. Engel for providing the Tulahuen strain TCT and Dr. R. Maignon (Liverpool School of Tropical Medicine, U.K.) for the c~ and fl tubulin probe.

References

1 Brener, Z. (1973) Biology of Trypanosoma cruzi. Annu. Rev. Microbiol. 27, 347-382.

2 De Souza, W. (1984) Cell biology of Trypanosoma cruzi. Int. Rev. Cytol. 86, 197-283.

3 Nogueira, N., Bianco, C. and Cohn, Z. (1975) Studies on the selective lysis and purification of Trypanosoma cruzi. J. Exp. Med. 142, 224-229.

4 Nogueira, N. and Cohn, Z. (1976) Trypanosoma cruzi: mechanism of entry and intracellular fate in mammalian cells. J. Exp. Med. 143, 1402-1410.

5 Contreras, V.T., Morel, C.M. and Goldenberg, S. (1985) Stage specific gene expression precedes morphological changes during Trypanosoma cruzi metacyclogenesis. Mol. Biochem. Parasitol. 14, 83-96.

6 Rangel-Aldao, R., Allende, O., Triana, F., Piras, R., Hen- riquez, D. and Piras, M. (1987) Possible role of cAMP in the differentiation of Trypanosoma cruzi. Mol. Biochem. Parasitol. 22, 39--43.

7 Gonzales-Perdomo, M., Romero, P. and Goldenberg, S. (1988) Cyclic AMP and adenylate cyclase activators stimu- late Trypanosoma cruzi differentiation. Exp. Parasitol. 66, 205-212.

8 Sullivan, J.J. (1982) Metacyclogenesis of Trypanosoma cruzi in vitro: a simplified procedure. Trans. R. Soc. Trop. Med. Hyg. 76, 300-302.

9 Alvarenga, N.J. and Brener, Z. (1979) Isolation of pure metacyclic trypomastigotes of Trypanosoma cruzi from tri- atomine bugs by use of a DEAE cellulose column. J. Par- asitol. 65, 814-815.

10 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rut- ter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299.

11 Okayama, H. and Berg, P. (1982) High-efficiency cloning of full-length cDNA. Moi. Cell. Biol. 2, 161-170.

12 Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269.

13 Sargent, T.D. and Dawid, I.B. (1983) Differential gene expression in the gastrula of Xenopus laevis. Science 222, 135-139.

14 Benton, W.D. and Davis, R.D. (1977) Screening Agtll recombinant clones by hybridization to single plaques in

situ. Science 196, 180-182. 15 Heath, S., Chance, M.L., Hommel, M. and Crampton,

J.M. (1986) Cloning of the gene coding for the immu- nodominant surface antigen of Leishmania donovani pro- mastigotes. Mol. Biochem. Parasitol. 23, 211-222.

16 Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13.

17 Wilkinson, M. (1988) RNA isolation: a mini-prep method. Nucleic Acids Res. 16, 10933.

18 Flower, R.J. (1974) Drugs which inhibit prostaglandin biosynthesis. Pharmacol. Rev. 26, 33~7.

19 Bairoch, A. (1989) Ph.D. Thesis, University of Geneva. 20 Teixeira, M.M.G. and Yoshida, N. (1986) Stage specific

surface antigens of metacyclic trypomastigotes of Try- panosoma cruzi identified by monoclonal antibodies. Mol. Biochem. Parasitol. 18, 271-282.

21 Martins, M.S., Hudson, L., Krettli, A.V., Cancado, J.R. and Brener, Z. (1985) Human and mouse sera recognise the same polypeptide associated with immunological resistance to Trypanosoma cruzi infection. Clin. Exp. Immunol. 61, 343-350.

22 Peterson, D.S., Wrightsman, R.A. and Manning, J.E. (1986) Cloning of a major surface antigen gene of Trypanosoma cruzi and identification of a nonapeptide repeat. Nature 322, 566-568.

23 Beard, C.A., Wrightsman, R.A. and Manning, J.E. (1988) Stage and strain specific expression of the tandemly re- peated 90 kDa surface antigen gene family in Trypanosoma cruzi. Mol. Biochem. Parasitol. 28, 227-234.

24 Takle, G.B., Young, A., Snary, D., Hudson, L. and Nicholls, S.C. (1989) Cloning and expression of a trypomastigote- specific 85-kilodalton surface antigen gene from Try- panosoma cruzi. Mol. Biochem. Parasitol. 37, 57~o4.

25 Goldenberg, S., Salles, J.M., Contreras, V.T., Lima Franco, M.P., Katzin, A.M., Colli, W. and Morel, C.M (1985) Char- acterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi. FEBS Lett. 180, 265-270.

26 Ucros, H., Granger, B. and Krassner, S.M. (1983) Try- panosoma cruzi: effect of pH on in vitro formation of meta- cyclic trypomastigotes. Acta Trop. 40, 105-112.

27 Contreras, V.T., Salles, J.M., Thomsa, N., Morel, C.M. and Goldenberg, S. (1985) In vitro differentiation of Try- panosoma cruzi under chemically defined conditions. Mol. Biochem. Parasitol. 16, 315-327.

28 Fernandes, J.F., Castellani, O. and Kimusa, E. (1969) Phys- iological events in the course of the growth and differentia- tion of Trypanosoma cruzi. Genetics 61, Suppl. 1, 213-226.

29 Rangel-Aldao, R., Triana, F., Fernandez, V., Comach, G., Abate, T. and Montoreano, R. (1988) Cyclic AMP as an inducer of the cell differentiation of Trypanosoma cruzi. Biochem. Int. 17, 337-344.

30 Montiminy, M.R., Sevarino, K.A., Wagner, J.A., Mandel, G. and Goodman, R.H. (1986) Identification of a cyclic- AMP-responsive element within the rat somatostatin gene. Proc. Natl. Acad. Sci. USA 83, 6682-6686.

31 Comb, M., Birnberg, N.C., Seasholtz, A., Herbert, E. and Goodman, H.M. (1986) A cyclic AMP and phorbol ester- inducible DNA element. Nature 323, 353-356.

32 Maurer, R.A. (1981) Transcriptional regulation of the pro- iactin gene by ergocryptine and cyclic AMP. Nature 294, 94-97.

33 Tsukada, T., Fink, J.S., Mandel, G. and Goodman, R.H. (1987) Identification of a region in the human vasoactive

intestinal polypeptide gene responsible for regulation by cyclic AMP. J. Biol. Chem. 262, 8743-8747.

34 Yun, Y. and Davis, R.L. (1989) Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP. Mol. Cell. Biol. 9, 692-700.

35 Montminy, M.R. and Bilezikjian, L.M. (1987) Binding of a nuclear protein to the cyclic AMP response element of the somatostatin gene. Nature 328, 175-178.

36 Yamamoto, K.K., Gonzalez, W.H. Biggs and Montminy, M.R. (1988) Phosphorylation-induced binding and tran- scriptional efficacy of nuclear factor CREB. Nature 334, 494-498.

37 Rangel-Aldao, R., Tovar, G. and Ledezma de Ruiz, M. (1983) The cAMP receptor protein of Trypanosoma cruzi.

141

J. Biol. Chem. 258, 6979-6983. 38 Ross, J. and Kobs, G. (1986) H4 histone messenger RNA

decay in cell-free extracts initiates at or near the Y terminus and proceeds 3' to 5'. J. Mol. Biol. 188, 579-593.

39 Strickler, J.E. and Patton, C.L. (1975) Adenosine Y,5'- monophosphate in reproducing and differentiated try- panosomes. Science 190, 1110-1112.

40Mancini, P.E. and Patton, C.L. (1981) Cyclic 3'5'- adenosine monophosphate levels during the developmen- tal cycle of Trypanosoma brucei in the rat. Mol. Biochem. Parasitol. 3, 19-31.

41 Walter, R.D., Base, E. and Ebert, F. (1978) Effect of cAMP on transformation and proliferation of Leishmania cells. Tropenmed. Parasitol. 29, 439-442.