Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

EIGNER & FOISSNER-MORPHOGENESIS IN AMPHISIELLID CILIATES 26 1

3 1. Wirnsberger, E., Foissner, W. & Adam, H. 1986. Biometric and motphogenetic comparison of the sibling species Stylonychia mytilus and S. lemnae, including a phylogenetic system for the oxytrichids (Ciliophora, Hypotrichida). Arch. Protistenkd., 132: 167-185.

32. Wirnsberger-Aescht, E., Foissner, W. & Foissner, I. 1989. Mor-

phogenesis and ultrastructure of the soil ciliate Engelmanniella rnobilis (Ciliophora, Hypotrichida). Europ. J. Protutol., 24:354-368.

Received 6-01-93, 1-03-94; accepted I-04-94

J. Euk. Microbrol., 41(3), 1994, pp. 261-267 0 1994 by the Society of Rotozoologsts

Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

MARIO STEINDEL,* EMMANUEL DIAS NETO,** CARLOS J. CARVALHO PINTO,* EDMUNDO C. GRISARD,* CARLA L. P. MENEZES,** SILVANE M. F. MURTA,** ANDREW J. G. SIMPSON** and ALVARO J. ROMANHA**.'

*Departmento de Microbiologia e Parasitologia Universidade Federal de Santa Catarina C. P. 476, 88040-900, Florianbpolis Santa Catarina. Brazil, and

**Centre de Pesquisas "RenC Rachou, " Av. August0 de Lima, 1715, 30190-002 Belo Horizonte MG. Brazil

ABSTRACT. Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co-infections with Trypanosoma cruzi, demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of 7'. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.

Supplementary key words. Genetic variation, isoenzymes, randomly amplified polymorphic DNA analysis (RAPD), phenogram, Trypanosoma cruzi, Trypanosoma rangeli.

RYPANOSOMA rangeti, first described by Tejera in 1920 T [ 171, is a trypanosome species that infects man in Central and South America where it can be found in mixed infections together with T. cruzi in both invertebrate and vertebrate hosts. While T. cruzi causes Chagas' disease, T. rangeli is considered nonpathogenic to man [4]. The high cross reactivity between T. cruzi and T. rangeli in different immunological assays is a major problem in the diagnosis of chronic Chagas' disease in those areas where both species are present [7].

The first unequivocal report of T. rangeli in Brazil was in the Amazon Basin [9]. More recently, the organism has also been isolated in the south ofthe country in the State ofSanta Catarina [ 151. Isoenzyme analysis ofthe single isolate reported from Santa Catarina suggested that it was distinct from T. rangeli from Honduras but was identified as being of the same species on the basis of its development in the insect vector, transmission to mice by bite, WGA lectin agglutination and complement lysis

In this communication we report a genetic comparison of eight strains of T. rangeli from Santa Catarina, Brazil; two from Venezuela; three from Colombia and three from Honduras by means of isoenzymes and randomly amplified polymorphic DNA (RAPD) [ 16, 20, 21, 221. RAPD are produced using the polymerase chain reaction (PCR) with arbitrarily chosen primers that are annealed to the template DNA (derived from the parasite) at low temperatures. The amplified DNA fragments represent anonymous regions distributed randomly throughout the genome of the parasite and provide a fingerprint of the isolate or species being studied. Phylogenetic relationships between organisms are estimated by measuring the number of amplified DNA fragments in common.

~ 5 1 .

I To whom correspondence should be addressed.

The results of both techniques confirm that T. rangeli from Brazil is genetically distinct from T. rangeli from Honduras. Colombia and Venezuela. In addition, the studies emphasize that RAPD represent a powerful means of identifying trypanosome strains and species in both single and mixed infections.

MATERIALS AND METHODS Parasites. Sixteen T. rangeli strains from different geograph-

ical regions were studied (Table I ) . The six strains (SC-66, SC-67, SC-68, SC-70, SC-7 1 and SC-72), isolated from Pansfrongylus megistus were found in mixed T. cruzi/T. rangeli infections. The purification of T. rangeli was achieved by infecting Rhod- nius domesticus and transmitting the parasite to mice via the bite of the infected insects. Two T. cruzi strains (SC-69 and SC- 73) were isolated from P. megistus from the same geographical region by xenoculture [2]. The parasites were identified as being T. cruzi or T. rangeli on the basis of their ability to be trans- mitted from triatomines to mice via the anterior route [ 151. Confirmatory studies on the course of infection in these hosts, isoenzyme patterns, WGA lectin agglutination as well as sensitivity of epimastigote culture forms to complement lysis were also undertaken as previously described [ 151.

All parasites were grown in LIT medium at 28" C, washed 3 times in PBS pH 7.4, collected by microcentrifugation at 13.000 g for 10 min at room temperature and stored at -70" C until use.

Isoenzyrne analysis. Pellets of parasite material were sub- mitted to osmotic lysis in an enzyme stabilizer (2 mM dithioth- reitol, 2 mM c- aminocaproic acid and 2 mM Na,-EDTA, pH 7.0) at 4" C, at the ratio 1: 1. The lysates were then centrifuged at 15,000 g for 1 h at 4" C and the supernatant, (enzymatic extract) cryopreserved in liquid nitrogen. Isoenzymes were separated by refrigerated, horizontal thin-layer starch gel electro-

262 J. EUK. MICROBIOL.. VOL. 41, NO. 3, MAY-JUNE 1994

Table 1 . Geographical origin of Trjpanosotna rangeli strains.

References Strain Host Method of isolation Geographical origin

HIGS, H-9. H-14 Horno sapiens Hemoculture Honduras Ref. [I] San Agostin (SA) Hotno sapiens Xenodiagnosis Colombia Ref. [I91 Choachi (CH), P- 19 R . prolixus Ref. [I21 Palma-2 (PL 2) R . prolixus Xenodiagnosis Venezuela a Macias (MA) Homo saprens Xenodiagnosis SC-58 . SC-6 1 E. dasyIhnx Hemoculture State of Santa Ref. [ 151 SC-66. SC-67 P. megislus Xenoculture Catarina, This paper SC-68. SC-70 P. megistus Xenoculture Brazil This paper SC-7 I , sc-72 P. megistus Xenoculture This paper

.* Samples sent by Dr. SPrvio Urdaneta-Morales from the Universidad Central de Venezuela.

phoresis. The following five enzymes were studied: alanine aminotransferase (ALAT) [E.C.2.6.1.2], aspartate aminotransferase (ASAT) [E.C.2.6. I . I ] , glucose phosphate isomerase (GPI) [E.C.5.3.1.9]. phosphoglucomutase (PGM) [E.C.2.7.5.1.] and malic enzyme (ME) [E.C. 1.1.1.40]. The running and development conditions were as previously described [3]. Four standard zymodemes of T. cruzi (ZI. Z2, ZB and ZC) were used for reference [3, lo]. DNA preparation. Extraction was undertaken essentially as

previously described for schistosomes [ 141. Briefly, the parasite pellet was resuspended in 50 mM Tris-HC1, 100 mM NaCI, 50 mM EDTA. 0.5% SDS, pH 8.0 and digested with 50 &mi proteinase K for 2 h at 37" C. Following phenol extraction and ethanol precipitation, the DNA was resuspended in 10 mM Tris- HCI, 1 mM EDTA pH 8.0 (TE) and digested with 1 m d m l RNase for 2 h at 37" C. Following a further round of phenol extraction and ethanol precipitation, the DNA was again resuspended in TE pH 8.0 and its concentration determined by comparison with known standards following electrophoresis in a 2% agarose gel stained with ethidium bromide.

Randomly amplified polymorphic DNA (RAPD). Each amplification reaction was done in a final volume of 10 pl containing 0.8 units of Taq DNA polymerase (Cenbiot RS, Brazil), 200 pM of each dNTP. 1.5 pM MgCI2, 50 mM KCI, 10 mM Tris-HCI, pH 8.5, together with 6.4 pmoles of primer (Table 2 ) and 1 .O ng of template DNA. This reaction mixture was overlaid with 20 ~l of mineral oil and, following an initial denaturation at 95" C for 5 min, was subjected to two cycles through the following temperature profile: 30" C for 2 rnin for annealing, 72" C for 1 rnin for extension and 30 s at 95" C for denaturation followed by 33 cycles where the annealing step was altered to 40" C. In the final cycle the extension step was for 5 min. Fol- lowing amplification, 3 fiI of the reaction was mixed with 0.6 pl of 6 x DNA sample buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) and subjected to electrophoresis through a 4% polyacrylamide gel. Gels were fixed with 10% ethanol/0.5% acetic acid for 20 rnin and the DNA bands vi- sualized by staining with 0.2% silver nitrate for 30 rnin and reduction with 0.75 M NaOH/O. 1 M formaldehyde for 10 rnin as previously described [ 1 I].

Table 2. Sequences of the primers used on the RAPD analysis.

Primer Sequence

3303 5'-TCACGATGCA-3' 3304 5'-GCACTGTCA-3' 3306 5'-AGCATCTGTT- 3' 3307 5'-AGTGCTACGT-3' Xgtl 1R 5'-TTGACACCAGACCAACTGGTAATG-3' Xgtl IF 5'-GGTGGCGACGACTCCTGGAGCCCG-3'

RAPD data analysis. From the data derived from four primers. a total of 138 DNA fragments (bands), that could be identified with confidence on the basis of their intensity and separation from other products on the gels, were considered for analysis. Phenetic trees based on band sharing between all possible pairs in an analysis group were constructed using the dice similarity coefficient S = 2a/2a + b + c where a = the number of bands shared between organism 1 and 2. b = the number of bands present in 1 but not 2 and c = the number of bands present in 2 but not 1 [S]. Data derived from this formula represent the percentage of common bands between every two individuals. They were plotted to establish a matrix of similarity that was then used for unweighted pair group method analysis (UPGMA). The phenon line marked on the UPGMA is the average of the similarities among the pairs and indicates the point of reference for dividing the organisms into separate groups 1131.

RESULTS Isoenzyme analysis using five enzymes was undertaken with

all the T. rangeli strains, including those isolated as naturally mixed infections with T. cruzi, together with four T. cruzistrains representative of zymodeme Z I , 22, ZB and ZC. Figure 1 a shows the results of one of the enzymes used (GPI) and Fig. 1 b shows a diagrammatic representation of the results. All the T. rangeli strains from Santa Catarina were isoenzymatically identical, one representative result is shown in the figures. The strains from Honduras, Colombia and Venezuela also formed a highly ho- mogeneous group, the only variation being the presence of an additional band for malic enzyme in the San Agostin (SA) strain from Colombia. The two groups showed distinct patterns for all enzymes except malic enzyme. We found that in all cases (six) that the T. cruzi strain that coinfected P. nipgistus with T. rangeli was zymodeme I (an example of the data is shown in the figures). The isoenzyme patterns of T. cruzi zymodeme 1 and T. rangeli from Santa Catarina were distinct in all cases thus allowing the identification of mixed infections using this methodology.

DNA extracted from all the strains was used as template with six arbitrarily selected primers under low stringency amplification conditions to produce RAPD. The strains from Santa Catarina were found to exhibit a low level of polymorphism as illustrated for primer Xgtl 1F (Fig. 2). The strains from Hon- duras, Colombia and Venezuela exhibited a higher degree of variation and were all quite distinct from the strains of Santa Catarina. Figure 3 illustrates the data obtained using primer XgtllF with all the strains from Honduras, Colombia and Ven- ezuela together with two of the strains from Santa Catarina selected at random for comparison.

The RAPD derived from 4 primers (3303, 3304, XgtllR and AgtllF) using the strains shown in Fig. 3 were used for the con-

Fig. 1. Isoenzyme patterns of the T. rangeli strains. a. Representative zymogram for the enzyme glucose phosphate isomerase (GPI) and b. A diagrammatic representation of the five enzymes studied. For enzyme and strain abbreviations refer to Materials and Methods and Table 1. Zl/SC-66 is a representative naturally occumng coinfection of T. cruzi and T. rangeli.

264 J. EUK. MICROBIOL., VOL. 41, NO. 3, MAY-JUNE 1994

Fig. 2. RAPD profile of T. rungelr strains from Santa Catarina State, Brazil, using the primer Xgt 1 1 F. For strain abbreviations refer to Table 1 . The lane on the left (M) shows molecular size markers.

struction of the phenogram shown in Fig. 4 as described in Materials and Methods. The phenon [ 131 line marked indicates the average divergence of all pairs and is used as a rational basis for the separation of distinct groups of organisms. Divergence prior to the position of the phenon line is taken as biologically significant. Thus, the data support the contention that the Santa

Catanna strains are distinct from those isolated in Honduras, Colombia and Venezuela. The validity of using RAPD for phylogenetic analysis is supported by the observation that the strains from Honduras, Colombia and Venezuela are also grouped ac- cording to their geographic origin.

As T. rungeli was found in mixed infections together with T.

Fig. 3. RAPD pattern of the T. rungeli strains from Santa Catarina State, Honduras, Colombia and Venezuela obtained with the primer Xgt 1 IF. For strain abbreviations refer to Table 1. The numbers on the left indicate the molecular size markers used.

STEINDEL ET AL.-ISOENZYME AND DNA ANALYSIS OF TRYPANOSOMA RANGELI STRAINS 265

-

0.5 0.6 0.7 0.8 0.9 1 .o

CH

P19 I

c

7 SA

h i MA

H8GS I

H14

Fig. 4. UPGMA phenogram of T. rangeli strains. The numbers on the horizontal scale were derived from the Dice similarity coefficient. The vertical bar represents the phenon line. For strain abbreviations refer to Table 1.

cruzi in six out of the eight strains from Santa Catarina, we examined the ability of PCR amplification to identify such cases. We found that the most useful primer for this purpose was 3306. Figure 5 illustrates the amplification of four distinct naturally mixed infections. In each case the RAPD profile of the naturally occumng mixed infection is the sum of the profiles of the com- ponent species. Mixtures of different proportions of DNA from the two species were tested and it was found that the presence of as little as 5% of T. rangeli DNA was readily detectable (Fig. 6).

DISCUSSION The results presented confirm and extend analysis of T. run-

geli found in southern Brazil in the State of Santa Catarina [ 151. Isolates from this region share common biological features, such as pattern of infection in the intermediate host, with T. rangeli strains from Colombia, Honduras and Venezuela. However, the isoenzyme and RAPD analyses shown indicate that the Santa Catarina strains are genetically distinct from isolates from the other regions included in the study.

The results obtained with isoenzymes were very clear cut in that all the strains from Santa Catarina had identical profiles which overlapped in only one instance with the other strains thus suggesting the existence of two separate groups of organisms. This is in agreement with previous studies which have also described differences in the isoenzyme profiles of T. rangeli strains originating from Brazil and Venezuela [ 181. The results obtained with RAPD, however, were more complex since this method is far more sensitive and a far greater number of loci are examined. Thus, the decision that the strains examined can

Fig. 5. RAPD profile of natural T. rangelilT. cruzi mixed infections using the primer 3306. Lanes 2 and 5 , T. cruzi zymodeme 1; lanes 3, 6, 8 and 9, naturally mixed parasites (T. cruzi/T. rangeli, SC-66, SC-67, SC-68, and SC-72); lanes 4 and 7, T. rangeli strains (SC-66 and SC-67) after purification; lane 10, negative control (no DNA added). The lane on the left (M) shows the molecular size markers. For details see Materials and Methods.

266 J. EUK. MICROBIOL., VOL. 41, NO. 3, MAY-JUNE 1994

Fig. 6 . The limit of sensitivity ofdetection of T. rangeli in a mixture of T. rangeli/T. cruzi by RAPD, using the primer 3306. The experiment was undertaken using different proportions of D N A from each species. The percentage of D N A from T. rangeli in the mixture was: lane 1 = 0%; lane 2 = 5%; lane 3 = 10%; lane 4 = 50%; lane 5 = 100%. The amount of DNA in each reaction was 1 ng. The 1: rangeli and T. cruzi strains used were H-9 and SC-14 (Zl) respectively. The lane on the left (M) shows the molecular size markers.

be meaningfully separated into two groups was made employing the method described by Sneath and Sokal 1131. This method used the position of the average level of similarity between all pairs in the study to draw the so called phenon line. Groups that are more divergent than this average are considered sig- nificantly different. Although separate groups were apparent for the organisms derived from Honduras, Colombia and Vene- zuela, the point of divergence between them was less than the average and only the difference between the Santa Catarina group and the others was found to satisfy the criterion imposed. Thus, the two methods of analysis led us to draw the same conclusion.

Others have also shown that conclusions drawn from experiments undertaken with isoenzymes and RAPD are identical [ 191 thus, in effect, validating the newer PCR based method. The latter offers some advantages over isoenzyme analysis in particular the very large number of loci that can be simulta- neously examined and the much smaller amount of biological material that is required ( 1 O3 parasites are sufficient for at least 10 PCR amplifications whereas lo8 parasites are required for a reasonably complete isoenzyme analysis). The analysis of much larger numbers of loci during RAPD analysis permits the de- tection of variation that is not apparent using isoenzymes as is the case here with the samples from Santa Catarina. In terms of cost, RAPD analysis compares favorably with isoenzyme analysis. The reagents required for a single amplification and analysis is in the range of US $2. Technically, RAPD analysis is extremely simple and immediately available to all investi- gators who have access to a thermal cycler since no previous DNA sequence data is required.

Inspection of the UPGMA phenogram shown indicates that the overall structure of the T. rangeli population is similar to

that of T. cruii with the existence of separate groups in this clonal organism and variation within groups being linked to geographical origin. In the case of T. rangefi, further studies are required to understand the possible biological significance of the existence of the two groups found. However, preliminary experiments have indicated that the genetic difference reported here is reflected in antigenic differences as judged by the use of monoclonal and polyclonal antibodies in both immunofluores- cence and immunoblotting studies (data not shown). A pressing question is posed by the fact that to date all the strains of T. rangeli that have been isolated from Santa Catarina have been from either a non-human vertebrate or invertebrate host. It has yet to be confirmed if they are also found in man.

All of the strains of T. rangeli isolated from P. niegistirs were mixed infections together with T. cruzi zymodeme 1, which suggests that in Santa Catarina an overlap in the sylvatic cycle of the 2 parasites occurs, possessing P. rnegisfus as a common intermediate host. Our results thus confirm those from Lucena and Vergetti [S] who found P. megistus naturally infected with T. rangeli in Brazil. The finding of mixed infections indicates that it is essential that methodologies are available to accurately identify the organisms in such cases. The divergence of the isoenzyme patterns of T. rangeli and T. cruzi permits this to be done. The more informative RAPD methodology can also identify such mixed populations and in the data reported we show that using primer 3306 a proportion as low as 5% of T. rangefi DNA can be readily detected. Experiments using DNA from four reference T. cruzi strains representing the four major zymodemes together with the two T. rangeli groups reported here showed that the test was viable with each combination (data not shown).

The finding of T. rangefi in the south of Brazil emphasizes that the range of this parasite is much greater than originally suspected, although its presence in the State of Tocantins, Cen- tral Brazil, was recently described based on morphological inspection of parasites found in naturally infected R. neglectus [6]. A complete survey will be necessary to fully explore the genetic variation of this parasite. The most important question to be answered is whether the strains from Santa Catarina represent an extreme of a continuous variation that correlates with the geographical distribution of the parasite. Furthermore, a precise mapping of the distribution of T. rangefi is essential to deter- mine the extent to which its presence must be taken into account in the diagnosis of Chagas’ disease.

ACKNOWLEDGMENTS We would like to thank Dr. Servio Urdaneta-Morales for

providing the Macias and Palma-2 T. rangeli strains and Pro- fessor Zigman Brener for his critical reading of the manuscript. The work reported received financial support from FIOCRUZ, CNPq and CAPES/PICD.

LITERATURE CITED I . Acosta, L.. Romanha, A. J., Cosenza, H. & Krettli, A. U. 1991.

Trypanosomatid isolates from Honduras: differentiation between Trypanosoma cruzi and Trypanosoma rangeli. A m . J . Trop. Med. Hyg., 44:676-683.

2. Bronfen, E., Rocha. F. S. A,, Machado, G . B. N., Perillo, M. M., Romanha, A. J . & Chiari, E. 1989. Isolamento de amostras de Trypanosoma cruzi por xenodiagnostico e hemocultura de pacientes na fase cr6nica de DoenGa de Chagas. Mein. Inst. Oswaldo Cruz, 841237- 240.

3. Carneiro, M., Chiari, E., GonGalves, A. M., da Silva Pereira, A. A., Morel. C. M. & Romanha, A. J. 1990. Changes in the isoenzyme and kinetoplast D N A patterns of Trypanosoma cruzi strains induced by maintenance in mice. Acta Tropica, 47:3545. 4. DAlessandro. A. 1976. Biology of Trypanosoma (Herpetosoma)

STEINDEL ET AL.-ISOENZYME AND DNA ANALYSIS OF TRYPANOSOMA RANGELI STRAINS 267

rangeli Tejera, 1920. In: Lumdsen. W. H. R. & Evans, D. A. (ed.), Biology of Kinetoplastida. Academic Press, London. 1:328-403.

5. Dice, L. R. 1945. Measures of the amount of ecological associ- ation between species. Ecology, 26:297-302.

6. Diotaiuti, L., Silveira, A. C., Elias, M. & Steindel, M. 1992. The possibility of occurrence of Trypanosoma rangeli in the State of To- cantins, Brazil. Mem. Inst. Oswaldo Cruz, 87:45.

7. Guhl, F.. Hudson, L., Marinkelle, C. J., Morgan, S. J. & Jaramillo, C. 1985. Antibody response to experimental Trypanosoma rangeli infection and its implications for immunodiagnosis of South American Trypanosomiasis. Acta Tropica, 42:311-3 18.

8. Lucena, D. T. & Vergetti, J. G. 1973. Infec@o natural de Pan- strongylus rnegistus (Burmeister, 1835) por Trypanosoma rangeli (Te- jera, 1920), no interior de Alagoas. Rev. Inst. Med. trop. Siio Paulo, 15:

9. Miles, M. A., Arias, J. R., Valente, S. A. S., Naiff, R. D., de Souza, A. A,, Povoa, M. M., Lima, J. A. N. &Cedillos, R. A. 1983. Vertebrate hosts and vectors of Trypanosoma rangeli in the Amazon Basin of Brazil. Am. J. Trop. Med. Hyg.. 32:1251-1259.

10. Miles, M. A., Lanhan, S. M., Souza, A. A. & Povoa, M. 1980. Further enzymic characters of Trypanosoma cruzi and their evaluation for strain identification. Trans. R. SOC. Trop. Med. Hyg., 74:221-237.

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Received 5-21- 93; accepted 1-4-94

J. Euk. Microbiol., 41(3), 1994, pp. 267-275 0 1994 by the Society of Rotozoologists

Accumulation of Telomerase RNA and Telomere Protein Transcripts during Telomere Synthesis in Euplotes

CAROLYN M. PRICE,' ANGELA K. ADAMS and JORIS R. VERMEESCH Department of Chemistry, University of Nebraska, Lincoln. Nebraska 68588

ABSTRACT. In the ciliate Euplotes crassus a complex series of developmental events lead to formation of a new macronucleus. Millions of telomeres are synthesized during this process. We have shown that transcript levels are tightly regulated throughout Euplotes conjugation and macronuclear development. Thus, expression of gene products needed for macronuclear development and telomere synthesis appears to be controlled at the level of RNA abundance. To learn more about the role played by telomerase and the Euplotes telomere protein during telomere synthesis, we have correlated changes in the abundance of telomerase RNA and telomere protein mRNA transcript with specific developmental events. Telomerase RNA levels increase steadily during the early stages of macronuclear development and reach a peak just after telomere addition. The telomere protein transcript rises and falls twice during conjugation and then rises again at the time of telomere addition. The increases in transcript levels during conjugation parallel micronuclear division suggesting that the telomere protein is synthesized at this time and hence may have a micronuclear function.

Supplementary key words. Euplotes crassus.

ELOMERIC D N A from many organisms consists of tan- T dem repeats of a short 5-8 base pair (bp) sequence that contains clusters of G-residues on the 3' strand [reviewed in 3 and 471. This repeated sequence D N A is synthesized by an RNA-containing enzyme called telomerase [4, 71. Telomerase can add telomeric repeats both to preexisting telomeres and to the ends of broken chromosomes during the process of chro- mosome healing [43, 441. The addition of telomeric repeats appears to be quite tightly regulated as telomere length only fluctuates by a few base pairs per generation [3]. Exactly how

To whom correspondence should be addressed.

this regulation is achieved is not well understood. However, it appears to be a complex process that involves not only telomerase but also telomere-binding proteins and perhaps other cel- lular components [3].

Euplotes crassus is a particularly suitable organism for study- ing both telomere length regulation and telomere synthesis be- cause it has large numbers of telomeres with a very well defined length, and it can be induced to undergo bulk de novo telomere synthesis [ 16,28, 351. Euplotes, like other ciliates. has two func- tionally and structurally distinct nuclei; the germline micronucleus, and the vegetative macronucleus [23]. In Euplotes. the micronucleus contains < 100 large chromosomes, whereas the macronucleus contains millions of gene-sized pieces of DNA

Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

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