restriction endonuclease banding and digestion resistant

CARYOLOGIA Vol. 53, no. 3-4: 235-243, 2000

Restriction endonuclease banding and digestion resistantheterochromatin in triatomines, genus PanstrongylusESTER TARTAROTTI* and MARIA TERCILIA VILELA DE AZEREDO-OLIVEIRA

Universidade Estadual Paulista - UNESP-IBILCE - Institute de Biociencias Letras e Ciencias Exatas, Departamento de Biologia. RuaCristovao Colombo, 2265, Jardim Nazareth - Sao Jose do Rio Preto - SP-Brasil - CEP: 15054.000

Abstract — The present work realized a comparative study in meiosis of two tri-atomines, Panstrongylus herreri and P. megistus, by cytogenetic techniques involving therestriction endonucleases Hae III and Alu I and C-banding. The system of sexchromosomes in Panstrongylus is of the XXX2Y type, and experiments corroboratedthe common origin hypothesis of the X chromosomes by fragmentation of single X. Inboth species the restriction endonucleases (RE) presented banding patterns in partsimilar to C-banding. However, in some early meiotic phases it was possible to verifydifferentiation of the heterochromatic pattern. This work sug-' gests that other elementsbesides presence of recognition sites, such as chromatin packing degree and DNA-protein interaction, act in RE results, since digestion patterns occur in earlyspermatogenesis. However, metaphase chromosomes were practically inaccessible to theendonucleases.

Key words: C-band, cytogenetic, restriction endonucleases, spermatogenesis, tri-atomines

INTRODUCTION

It is estimated that about 20 million indi-viduals with Chagas' disease exist in the endemicareas of the Americas (CARVALHO et al. 1993; DIAS1993; KIRCHHOFF 1993; CARDOSO et al. 1995; GRIJALVAet al 1995; LUQUETTI 1995; GUHL and SCHOFIELD1996; ALTCLAS et al. 1996; REICHE et al. 1996;COLLI et al. 1998). There occur approximately50,000 deaths a year as the result of Trypanosomacruzi infection (ALTCLAS et al. 1996; GALEL andKIRCHHOFF 1996). In Brazil about four millionpeople are chagasic (COLLI et al. 1998), and10,000 to 20,000 new cases appear annually(REICHE et al. 1996).

Triatomine populations have wide geo-graphical distribution, being found mainly inCentral and South America. The domesticationof these insects is a complex phenomenon. Sev-eral hypotheses have been suggested for this do-

* Corresponding author: fax ++55 17 2212390; e-mail:[email protected]

mestication, such as genetic mutation and laterhome adaptation, environmental and climaticconditions and food and shelter availability(STEINDEL et al. 1994).

The means of transmission of Chagas' de-sease can be by deposition of triatomine feces(NEVES 1988), congenital infection (BRABIN 1992;LUQUETTI 1994) and blood transfusion(WANDERLEY et al. 1992). Trypanosoma cruzi canalso be transmitted during organ transplants, suchas kidneys and heart (NEVES 1988).

There are approximately 100 triatomine spe-cies, all capable of harboring T. cruzi (SILVEIRA1983). Most of the triatominae subfamily mem-bers have 10 autosome pairs and one pair of sexchromosomes or multiple sex chromosomes.The modal number of chromosomes in triatom-ines is 22 (20+XY) (UESHIMA 1966). The multiple Xchromosomes originated by the simplefragmentation of an original X (UESHIMA 1979).

Cytogenetic studies of triatomines have re-vealed that their chromosomes possess a diffusekinetochore, that is, distributed along of thechromosome and not located in a centromericarea, and an unusual form of meiosis, in that

236 TARTAROTTI and AZEREDO-OLIVEIRA

segregation of the sex chromosomes is pos-treductional (DE VAIO et al. 1985). A combinedstudy using conventional cytogenetic techniquesand restriction endonucleases (REs) contributesgreatly in the analysis of chromatin heterogeneity(GOSALVEZ et al. 1987). The principalendonucleases applied in the study of insectcytogenetic are Alu I, Hae III, Hind III and HinfI. This work realized a comparative study of P.herreri and P. megistus by cytogeneticstechniques of banding with the restriction en-donucleases Hae III and Alu I and C-banding.

MATERIALS AND METHODS

The species analyzed were Panstrongylus megistus(2n=18+X1X2Y) and P. herreri (2n=20+X1X2Y)(order Heteroptera, family Reduviidae, sub-familyTriatominae) (LENT et al. 1979; MANNA 1995). Theorgans studied were testes of young adult males. Theinsects were provided by the Insectary of the SpecialHealth Service, Araraquara (SESA), which is run bythe Department of Epidemiology, Faculty of PublicHealth, Sao Paulo (SP-Brasil). The techniques appliedwere C-banding, according to SUMNER (1990), andbanding with the restriction endonucleases Alu I (AG |CT) and Hae III (GG j CC), Sigma ChemicalCompany. 10 units of endonuclease were used for100 �l of buffer (lOmM of Tris/HCl, pH 7,5; 50 mMof NaCl; 10 mM of MgCl2; and 1 ditiotreitol mM).The incubation period ranged from 16-18 hours at37°C. Digestion controls were done only with buffer.The preparations were stained with Giemsa 3 % for 20min in phosphate buffer (pH 6,8). Thephotomicrographs were made using a Zeiss-Jenevalphotomicroscope.

RESULTS

The present work analyzed meiosis in thetriatomines Panstrongylus megistus and P. her-reri by digestion with the endonucleases Hae IIIand Alu I, and the results were compared withC-banding. With the endonuclease Alu I thepolyploid nuclei of the nutritive cells of the tu-bule wall, in both species, presented a patternsimilar to that of C-banding, with dispersechromatin and only one heterocromatic corpuscle(figure la C-band and 1c Alu I; figure 3a C-bandand 3c Alu I). Hae III presented a pattern similarto C-banding in P. herreri (figure 3a C-bandand 3b Hae III); however in P. megistus

Fig. 1 — Testicular tubules of P. megistus. (a) Polyploid nuclei.C-banding, showing a single heterochromatic corpuscle. (b)Hae III. Note halo around corpuscle and nuclear chroma-tinwith digestion, (c) Alu I. Note nuclear chromatin and haloaround corpuscle, heterochromatin without digest. Scale bar, 10�m.

the nuclei presented only a heterochromaticcorpuscle, surrounded by an enzymatic diges-tion halo. The chromatin showed signs of diges-tion by being less compact compared to C-banding (figure la C-band; Ib Hae III).

In prophase I, with Alu I, P. megistus pre-sented heterochromatin with signs of longitudinaldigestion in some autosomes (figure 2 a C-band, 2c Alu I). In P. herreri, Alu I presented

RESTRICTION ENDONUCLEASES AND C-BANDING IN PANSTRONGYLUS 237

Fig. 2 — Testicular tubules of P. megistus. (a) Early prophase I (confused stage). C-banding. Arrow indicate heterochromatic sexchromosomes, (b) Hae III, note dispersed heterochromatin along the autosomes. (c) Alu I. Note autosomal chromatin showing signsof longitudinal digestion (arrows), (d) Pachytene. C-banding, note the heterochromatin spread along of the chromosomes, (e) Hae III.Arrows indicate autosomes showing a longitudinal pattern of enzymatic digestion, (f) Alu I. Arrows indicate heterochromatin presentin terminal areas of the chromosomes, (g) Metaphases of the second meiotic division. C-banding. (h) Hae III. (i) Alu I. In this meioticstage the heterochromatin becames resistant to endonucleases and C-banding. Scale bar, 10 �m.

a pattern similar to C-banding, that is, hetero-chromatic blocks located in terminal portions ofthe autosomes and heterochromatic sexchromosomes (figure 4a,d C-band, 4c,f Alu I).In diplotene the heterochromosomes are alsoobserved quite condensed with both C-bandingand Alu I (figure 4j C-band, 4m Alu I). With REHae III P. megistus presented dispersed hetero-chromatin along the autosomes, similar to C-banding (figure 2a C-band, 2b Hae III). In P.herreri the heterochromatin behaved in a dis-persed way along the autosomes, and the sexchromosomes, probably Xs, became digestedexhibiting a pattern different from that of C-banding (figure 4b,e Hae III). In diplotene

this digestion pattern can also be observed in theX chromosomes (figure 41 Hae III).

In pachytene with Hae III both species pre-sented a longitudinal enzymatic pattern of di-gestion of the autosomes (figure 2d C-band, 2eHae III P. megistus; figure 4g C-band, 4h HaeIII P. herreri). Treatment with Alu I produced apattern similar to C-banding in P. herreri, withtelomeric heterochromatin (figure 4g C-band, 4iAlu I P. herreri), and in P. megistus heterochro-matic blocks were observed in the telomeric areaswith longitudinal digestion of the autosomes(figure 2d C-band, 2f Alu I P. megistus).

In meiotic metaphase, after treatment withthe two restriction endonucleases and C-band-


ing, the chromosomes of both species becamequite condensed and without bands (figure 2g C-band, 2h Hae III, 2iAlu I P. megistus).

DISCUSSION

The sex chromosome mechanism in sub-family Triatominae includes systems of XY upto X1X2X3Y in the male. Systems of multiplesex chromosomes are more common in Heter-optera than in other insects, due to the holocen-tric nature of their chromosomes. When multi plesex chromosomes occur in insects, such asOrthoptera and Diptera, there is a tendency for adecrease in autosome number, due to chro-mosome fusion (UESHIMA 1966). In Heteropterathis type of relationship does not exist. Thediscovery of the holocentric nature of the chro-mosomes in these insects is based on morpho-logical behavior with experimental verificationafter breaking with ionic radiation. For exam ple,chromosome fragments experimentally inducedby X-ray continued to function mitoti-cally(HUGHES-SCHRADER and SCHRADER 1961).

In the two triatomine species analyzed, thesex chromosomes presented a characteristicpattern of coloration in early meiotic stages.Thus, in P. megistus the heterochromosomeswere totally heterochromatic after digestion withHae III and Alu I and C-banding. In P. herreri,the results with Alu I and C-banding werecoincident, that is, sex chromosomes totallyheterochromatic. With Hae III, digestion ofboth X chromosomes was observed. Theidentical X chromosome behaviour indicatesthat the heterochromatin present in these chro-mosomes is very similar, corroborating the hy-pothesis that X chromosomes originated byfragmentation of a single original X.

The heterochromatic heterogeneity of the Xchromosomes has also been studied in parthe-nogenetic females of the Homopteran, Megouravicia, by C-banding and digestion with the REsDde I, Dra I, Tru 91 and Cfo I, and showed thatin M. vicia C-bands occurred almost exclusivelyin the X chromosome. Also, in situ digestionwith restriction endonucleases produced longi-tudinal differentiation exclusively in the pair ofX chromosomes (MANICARDI et al. 1996). Re-striction endonucleases can also be useful forthe study of B chromosome origin. The hetero-

Fig. 3 — Testicular tubules of P. herreri. (a) Polyploid nuclei.C-banding showing a single heterochromatic corpuscle, (b)Hae III. Note the same chromatin pattern as C-banding. (c)AluI. Observe chromatin pattern similar to C-banding. Scale bar,10 �m.


chromatin in the grasshopper Eyprepocnemisplorans was analyzed by digestion with differentrestriction endonucleases and C-banding. Thepattern produced by the endonucleases Mho I,Msp I, Dde I, Bamb HI, Rsa I, Hae III, Alu I andHinf I was similar to the C-banding pattern, andthe endonucleases Hin pl, Scr FI, Sau 96A andTaq I removed part of the constitutive hetero-chromatin. The results indicated that the evolu-tionary process generated heterogeneity withindifferent chromatin fractions, and that the ma-terial that originates B-chromosomes involvesreorganization and amplification of autosomicmaterial (LOPEZ-FERNANDEZ et al. 1992).

In meiotic prophase I,of both P. megistusand P. herreri, the endonuclease Alu I presenteda pattern similar to C-banding. Thus, in P.herreri the heterochromatic blocks were locatedin the terminal regions of the autosomes, and thesex chromosomes were heterochromatic. In P.megistus, although some autosomes presentedregions of digestion, in general, heterochromaticblocks were dispersed along the chromosomes,and the heterochromosomes remainedheterochromatic. The chromosomes of themosquito Culiseta longiareolata submitted todigestion with the REs Alu I, Hha I, Hae III,Hpa II, Msp I, Dde I, Hinfl and Bam HII alsopresented heterochromatic areas protected fromendonuclease digestion with a pattern similar toC-banding. Only Mbo I caused longitudinaldifferentiation in the chromosomes (MARCHI andMEZZANOTTE 1988).

Some organisms such as the bivalve Mytilusgalloprovincialis, offer examples of a chromo-some differentiation pattern distinct from thatobserved by C-banding. C-banding revealed asmall amount of telomeric and interspersedconstitutive heterochromatin along the chro-mosome arms. The REs Alu I, Hae III, Dpn I,Msp I, and Hinf I revealed patterns of specificbanding and allowed the detection of hetero-chromatin heterogeneity (MARTINEZ-LAGE et al.1994). In Orthoptera, all the chromosomes ofArcyptera tornosi analyzed by C-banding re-vealed small centric bands, and some bands oc-curred distally. Banding with the endonucleasesAlu I, Hae III and Hinf I revealed chromosomedifferentiation similar to C-banding (GOSALVEZet al. 1987).

In pachytene of both Panstrongylus species,in situ digestion with the endonuclease Hae IIIpresented a longitudinal banding pattern in

some autosomes which indicates the presence ofGC rich areas, and the occurrence of enzymaticdigestion in early meiotic stages. Alu I produceda pattern similar to C-banding in P. herreri, andin P. megistus the heterochromatin was observedmainly in the telomeric areas.The holocentricchromosomes of the Homopterans Sitobionavenae and Schizaphis graminum were alsoanalyzed by C-banding and REs banding usingAlu I and Hae III. In the first species RE did notpromote different banding. In S. graminum, HaeIII produced terminal bands in two largechromosomes, similar those found in C-banding.Digestion with Alu I presented one of the largerchromosomes with interspersed banding (CELISet al. 1997). In the polytenic chromosomes ofDrosophila virilis treated with Alu I and Hae III,both enzymes digested the chromosomal DNA,except for some areas that contained repeatedDNA (MEZZANOTTE et al. 1987).

In the chromosomes of the OrthopteransDociostaurus jagoi and D. genei the REs Mbo Iand Sau 3A digested extensively the centro-meric area of both species, and heterochromaticsupernumerary segments present in D. geneiwere digested by Alu I. In both species, allchromosomes presented pericentromeric C-banding. Most of the enzymes applied pro ducedpatterns similar to C-banding. However, Hae IIIand Hpa II digested certain pericentromeric bandsin D. genei (RODRIGUES INIGO et al. 1993). TheREs Alu I, Dde I, Hae III, Hinfl, Hpa II, Mbo I,Msp I and Taq I produced longitudinaldifferentiation in the chromosome complementof the Orthopteran Oedipoda ger-manica(LOPEZ-FERNANDEZ et al. 1989). In Ten-ebriomolitor the constitutive heterochromatinrevealed by C-banding occupies extensive blocksin the pericentromeric areas of all chromosomes,and the Y chromosome was heterochromatic.The endonuclease Alu I provided a patternsimilar to C-banding, while Eco RI induced areverse banding pattern (JUAN et al. 1990).

Evidence suggesting that the resistance ofsome C-banding to restriction endonucleases isdue to lack of cleavage sites is not very prob-able. A series of factors influence enzymatic di-gestion, such as degree of chromatin packing,DNA-protein interaction, size of DNA frag mentgenerated, enzymatic conformation andmethylation. For example, the activity of Mbo I


Fig. 4 — Testicular tubules of P. herreri. (a) Prophase I (confused stage). C-banding Arrows indicate heterochromatic points in terminalchromosome region, (b) Hae III. Arrow indicates sex chromosomes, (c) Alu I. Note telomeric heterochromatic areas, (d) Meioticprophase. C-banding I, earlier stage. Arrow indicate sex chromosomes, (e) Hae III. Note that the X chromosomes are digested (arrowhead), (f) Alu I. Observe the heterochromatic sex chromosomes, (g) Pachytene. C-banding. Note heterochromatic terminal areas, (h)Hae III. Arrows indicate autosomal areas digested by endonuclease. (i) Alu I. Note that the terminal heterochromatic areas coincidewith C-banding. (j) Diplotene. C-banding. Arrow indicate the heterochromatic sex chromosomes. (1) Hae III. Observe that the Xchromosomes are digested (arrowhead), (m) Alu I. Arrow mark the heterochromatic sex chromosomes. Scale bar, 10�m.


is inhibited by adenine methylation in theGATC sequence, and cytosine methylation in-hibits Sau 3 A (GOSALVEZ et al. 1989).

Results of the present work indicate thatother factors other than the presence of endo-nuclease recognition sites act in the results ob-tained after treatment with REs. The two tri-atomines species analyzed with Hae III and Alu Ipresented digestion patterns in early meiosis,that is, the confused phase, pachytene anddiplotene, and also in the polyploid nuclei of P.megistus. However the metaphase chromosomes,whose degree of chromatin packing is high,became practically inaccessible to endo-nucleaseaction. Thus chromatin packing degree, sometype of DNA-protein interaction or other factorsmust have influenced the results, impedingendonuclease action or turning the DNA an areaof difficult extraction, even after cleavage. InAllium subvillosum the heterochro-matic regionswere practically resistant to enzymatic digestion,and digestion of these areas with the REs HaeIII and Msp I occurred after treatment withproteolitic or nucleolitic agents, which indicatespresence of proteic interaction interfering in REaction (FERNANDEZ-PIQUERAS et al. 1992).

Some authors believe that the DNA of allchromosome regions is equally accessible to re-striction endonucleases (MILLER et al. 1984;SENTIS 1988; LOPES-FERNANDEZ et al. 1988). BIANCHI etal. (1985) reported that the restrictionendonucleases Alu I, Mho I and Hae III inducedsimilar bands in the G1 phase pre-condensedchromosomes when compared with the bandsinduced in the mitotic chromosomes. Theseauthors affirmed that chromatin organization hasno role in the mechanism of chromosome bandingby REs. However, GOSALVEZ et al. (1989) believethat the data are not conclusive. Differentorganizations of distinct chromatin classes canaffect the action of endonucleases onchromosomes (PETITPIERRE et al. 1996).

RE activity on chromosomes is not only re-lated to the presence or absence of restrictionsites in a specific chromosome area. For exam ple,the endonuclease Eco RII, although capable ofcleaving extensively purified mouse satelliteDNA (SOUTHER 1975), does not attack satelliteheterochromatin in cytological preparations ofmouse chromosomes. Also, LICA and HAMKALO(1983) demonstrated that the activity of the en-donucleases Alu I and Eco RII in metaphase

cells is possibly affected by the high degree ofcondensation of centric heterochromatin, whileMEZZANOTTE et al. (1983) postulated that theactivity of several restriction enzymes on humanchromosomes depends on chromatin organiza-tion.

Human and mouse chromosomes treatedwith isoschizomers Mho I/Sau 3 A and Eco RII/Bst NI presented different results, indicatingthat factors other than DNA base compositioncan affect in situ digestion by restriction endo-nucleases, and that enzyme structure can be afactor. The isoschizomers Mbo I and Sau 3 Aproduced the same effect on the chromosomesof the grasshopper Oedipoda germanic. This in-dicates that differences in chromatin structureof different species can be important in deter-mining the activity of endonucleases on eu-karyotic chromosomes (GOSALVEZ et al. 1989).

Little is known of the role of endonucleaserestriction structure as concerns DNA diges-tion. Differences such as molecular weight andRE amino acid composition can explain the re-sults of the activity of isoschizomers on chromo-somes (GOSALVEZ et al. 1989). In this mannerrestriction endonuclease structure, as well asthe interaction between the restriction endonu-clease molecule and chromatin structure or-ganization, are factors that should be consid-ered in any conclusions about the effect of REson in situ digestion.

Acknowledgements — The authors are indebtedto Dr. Jose M. Scares Barata, Director of Insectary(Araraquara, SP), Epidemiology Department, PublicHealth Faculty (Sao Paulo, SP), and to Joao LuisMolina Gil and Bento Gregorio de Jesus, technicians ofthe Insectary for providing the insects. Thanks are dueto Dr. Marlene K. Hosaki Kobayashi for laboratorytraining, Dr.James Robert Coleman for reading themanuscript and to Maria do Carmo Junqueira forreading references. The support of CAPES andFAPESP is gratefully acknowledged.

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Received 31 July 2000; accepted 11 October 2000

restriction endonuclease banding and digestion resistant

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