Molecular Microbiology (1994) 13(6), 103% 1043

Molecular characterization and transcription of thehistone H2B gene from the protozoan parasiteTrypanosoma cruzi

Jose A. Garcia-Salcedo,’ Jose L. Oliver,’Roberto P. Stock’ and Antonio Gonzalez’*‘Instifufo de Parasitología y Biomedicina, ConsejoSuperior de lnvestigaciones Cientificas, Calle Ventanilla,7 1, 18001 Granada. Spain.Departamento de Genética Facultad de Ciencias,Universidad de Granada, 18071 Granada, Spain.

Summary

The structure, genomic organization and transcrlptlonof the gene encoding hlstone H2B In the protozoanparasite Trypansosoma cruzl have been studied. Thisgene consists of a 746~nucleotide unit, tandemlyrepeated at least 18 times in each of two clusters.DNA probes corresponding to hlstones H2B and H3hybridized to different chromosomes revealing thatthe genes coding for these two hlstones are not physl-cally linked in the genome of r . cruzl. The primarytranscription product of the H2B gene Is processedby trans-splicing and polyadenylatlon. lnhibltlon ofDNA synthesis with aphidicolin resulted in the reduc-tlon of histone H2B mRNA to undetectable levels inabout two hours, suggesting that Its abundance isregulated throughout the cell cycle as it occurs inother eukaryotes. in addition, a concomitant inhibitionof translation by cyclohexfmide reverted this effectindicating that de novo protein synthesis is requiredfor RNA instability. Hlstone mRNA abundance wasdependent on the life-cycle stage of r. cruzl: abun-dant in amastigotes and epimastlgotes, the dividingforms in the host cell and the Insect vector, respec-tively, while undetected in trypomastigotes, the para-site’s nondlvidlng life stage.

Introduction

Trypanosoma cruzi is a haemoflagellated protozoanbelonging to the order Kinetoplastidae, and is the causa-tive agent of Chagas’ disease, which afflicts millions ofpeople in the American continent (World Health Organiza-tion 1989). Its life cycle Includes several developmental

Receivedd 28 February, 1994: revised 6 June, 1994: accepted 8 June,1994. ‘For correspondence. Tel. (58) 203802; Fax (58) 203323.

stages (De Souza, 1984). Amastigotes and epimastigotesare replicative forms that are found in the cytoplasm ofmammalian cells and in the midgut of invertebrate hosts,respectively. Trypomastigotes are nondividing formsresponsible for the infectivity 01 the parasite towards awide range of mammalian cells. Like all members of thefamily Trypanosomatidae, 7. cruzi exhibits a number ofmolecular and biochemical properties not, or rarely,found in other eukaryotes. Polycistronic transcription(Gonzalez ef al., 1985; Muhich el al., 1988) trans-splicing(Murphy ef al., 1986) or mitochondrial RNA editing(Simpson el al., 1987) are phenomena that, together withorganelles such as glycosomes and a unique kinetoplast-containing mitochondrion, reflect the ancient evolutionarydivergence of these organisms.

Histones are basic proteins that are essential com-ponents of the structural framework that packages DNAin the nucleus of eukaryotic cells (Lewin, 1980). Fourtypes of histones, H2A, H2B, H3 and H4, form an octamercore around which DNA is coiled to establish the funda-mental unit of chromatin, the nucleosome (Simpson,1978). A fifth histone protein, Hl, binds to the linker regionbetween nucleosomes. Histones can undergo post-translational, covalent modifications (Ajiro et al., 1985;Busch and Goldknopf, 1981; De Lange et al., 1973;Doenecke and Gallwitz, 1982; Levy-Wilsson, 1983). Theresulting heterogeneity of histones is considered to beimportant for differences in chromatin structure which inturn may influence gene expression. In trypanosomatids,mitosis is characterized by the lack of condensed chromo-somes (Solari et a/., 1985). Few studies have been carriedout on the chromatin structure and composition in theseorganisms, most of them in 7. cruzi (Solari et al., 1985;Rubio et a/., 1980). The nucleosomes have beendescribed: they consist of a DNA repeating unit of about200 bp and a set of core histones, which resemble thosefound in higher eukatyotes. Four proteins seem to berepresented in the nucleosomal core histones of 7. cruzi(Astolfi el al., 1980; Toro and Galanti. 1990), Trypano-soma brucei (Bender, 1992), Trypanosoma lewisi(Elpidina et al., 1979) and Crithidia fasciculata (Duschakand Cazzulo, 1990). Their overall amino acid compositionis similar to that of the histones from lower eukaryotes.The postulated absence of histone Hl in Trypanosomati-dae (Rubio et a/., 1980) is in contradiction to the recent

1034 J. A. Garcia-Salcedo. J. L. Oliver, R. P. Stock and A. Gonzalez

descnption of proteins that are consldered to be a sub-species of histone t-l1 in T. cruzi (Toro and Galanti.1990) and in C. fasciculata (Duschak and Cazzulo, 1990).

The structures of genes and mRNAs encoding histoneshave been analysed in many organisms (Maxson el al.,1983). Histone genes are often clustered in genomicDNA but do not exhibit any single conserved topology.Their transcripts are not polyadenylated in higher eukary-otes but contain a conserved stem-loop structure at their3’ end (Wells and Kedes, 1986). On the other hand,mRNAs for ‘basal’ histones (a less abundant class of his-tones in eukaryotes) (Mannironi et al., 1989) and for thehistones from lower eukaryotes (fungi, ciliates and yeast)are polyadenylated and do not contain stem-loop struc-tures (Osley, 1991). Typically, histone protein synthesisand mRNA abundance are coupled to the cell cycle: his-tone biosynthesis is restricted to the S phase, occurringsimultaneously with DNA synthesis and chromosomereplication (Marashi et al., 1982). In general, three path-ways regulate the production of histone mRNA in thecell cycle of higher eukaryotes (Baumbach et a/., 1987;Harris et al., 1991; Heintz el al.. 1983; Sittman et a/.,1983). The first one regulates transcription at the Gl-Sphase boundary: as cells enter S phase, nascent histonemRNA synthesis increases three- to fivefold. The tworemaining pathways act post-transcriptionally. One actsin the cytoplasm to control the half-life of histonemRNAs: in the absence of DNA synthesis, histonemRNAs are rapidly degraded. The second pathway actsin the nucleus to process histone pre-mRNAs (Stauberand Schumperii, 1988). In higher eukaryotes, the stem-loop at the 3’-untranslated end is required for both proces-sing and degradation of histone RNAs (Bimstiel el a/.,1985; Pandey and Marzluff, 1987). The minor polyadenyl-ated ‘basal’ histone mRNAs are not regulated during thecell cycle (Wu and Banner, 1981) and are not sensitiveto the inhibition of DNA synthesis (Sittman et a/., 1983).In contrast, polyadenylated histone mRNAs in yeast cellsare cell-cycle regulated and destabilized in the absenceof DNA synthesis (Lycan et al., 1987; Xu et a/., 1990).

To the best of our knowledge, three histone genes havebeen characterized to date from kinetoplastid protozoa: thegenes coding for histone H2B from Leishmania enrieiiii(Genske et a/., 1991) and for histone H2A from Leish-mania donovani infanturn (Soto el al., 1992) and T. cruzi(Puefta el al., 1994). Their transcription products are poly-adenylatec!. However, it is reported (Genske et a/., 1991)that inhibition of DNA synthesis in L. enrieftii does notdecrease the relative level of histone H2B mRNA, suggest-ing that its abundance is not coupled to the Leishmania cellcycle.

We report here the cloning, molecular characterizationand genomic organization of the gene coding for histoneH2B in T. cmzi and show that its transcription product is

polyadenylated. InhibItIon of DNA synthesis results indecreasing histone H2B mRNA abundance to a very lowlevel, suggesting that histone gene expression is coupledto the cell cycle in Trypanosoma. In accordance with this,while the H2B mRNA is readily detected in the life formsable to divide (epimastlgotes in the insect vector andamastigotes in the mammal host), it can be barelydetected in the non-dividing trypomastigotes.

Results

Cloning of a T. cruzi histone HZB cDNA fragment

Taking advantage of the fact that all mRNAs from T. cruzistart with the same 39 nucleotides (the spliced leader,added by trans-splicing to the primary transcription pro-duct of any gene) (De Lange et al., 1984), we cloned the5’ ends of transcripts of interest by synthesizing cDNAusing reverse transcriptase and primers specific for parti-cular sequences, and then amplifying the cDNA bythe polymerase chain reaction (PCR), using the sameprimer and a second one corresponding to the splicedleader sequence. Owing to the low-stringency conditionsin the priming of the reverse transcriptase reaction andbecause in many instances we utilized degenerate firstprimers, it was not unusual to end up with more than oneamplified product (as seen by agarose gel electrophor-esis). By characterizing these bands we have obtaineduseful information on the 5’ ends of numerous T. cruzitranscripts (J. A. Garcia-Salcedo et a/., in preparation). Inan experiment using the oligonucleotide described inthe Experimental procedures, a 200 bp band was ampli-fied, isolated, cloned in an EcoRV-linearized pBSKS-(plasmid p41 EC) and sequenced. The amino-acid-derivedsequence from this cDNA, starting at the first ATG down-stream of the spliced leader, showed strong homology toH2B histones in protein databases. As shown below, thiscDNA fragment does correspond to T. cruzi histone H2B(nucleotides 78-258 plus 30 nucleotides of the splicedleader).

Genomic organization of the T. cruzi histone HZBgene

The genomic organization of T. cruzi histone H2B genewas studied by Southern blotting and chromosomal blot-ting of genomic DNA using the cDNA probe. Figure 1Ashows the bands obtained in a blot of DNA digested witha number of restriction enzymes. This pattern concurswith that of a tandemly repeated gene unit of about750 bp. Almost all copies of the gene contain one site forXbal and Sacl, most have two Pstl sites and there is anNcol site that is polymorphic among the repeating copiesand/or alleles. Finally, the enzymes Kpnl and Hindlll do

1034 J. A. Garcia-Salcedo. J. L. Oliver, R. P. Stock and A. Gonztilez

descnptton of proteins that are consldered to be a sub-species of histone t-i1 in T. cruz~ (Tore and GalantI.1990) and in C. fasoculata (Duschak and Cazzulo, 1990).

The structures of genes and mRNAs encoding histoneshave been analysed in many organisms (Maxson et al..1983). Histone genes are often clustered in genomicDNA but do not exhibit any single conserved topology.Their transcripts are not polyadenylated in higher eukary-otes but contain a conserved stem-loop structure at their3’ end (Wells and Kedes. 1986). On the other hand,mRNAs for ‘basal’ histones (a less abundant class of his-tones in eukaryotes) (Mannironi et al.. 1989) and for thehistones from lower eukaryotes (fungi, ciliates and yeast)are polyadenylated and do not contain stem-loop struc-tures (Osley, 1991). Typically, histone protein synthesisand mRNA abundance are coupled to the cell cycle: his-tone biosynthesis is restricted to the S phase, occurringsimultaneously with DNA synthesis and chromosomereplication (Marashi el al., 1982). In general, three path-ways regulate the production of histone mRNA in thecell cycle of higher eukaryotes (Baumbach et a/., 1987;Harris ef a/., 1991; Heir& et al.. 1983; Sittman ef al.,1983). The first one regulates transcription at the Gl-Sphase boundary: as cells enter S phase, nascent histonemRNA synthesis increases three- to fivefold. The tworemaining pathways act post-transcriptionally. One actsin the cytoplasm to control the half-life of histonemRNAs: in the absence of DNA synthesis, histonemRNAs are rapidly degraded. The second pathway actsin the nucleus to process histone pre-mRNAs (Stauberand Schumperli, 1988). In higher eukaryotes, the stem-loop at the 3’-untranslated end is required for both proces-sing and degradation of histone RNAs (Bimstiel et al.,1985; Pandey and Marzluff, 1987). The minor polyadenyl-ated ‘basal’ histone mRNAs are not regulated during thecell cycle (Wu and Bonner, 1981) and are not sensitiveto the inhibition of DNA synthesis (Sittman et a/., 1983).In contrast, polyadenylated histone mANAs in yeast cellsare cell-cycle regulated and destabilized in the absenceof DNA synthesis (Lycan et al., 1987; Xu et a/., 1990).

To the best of our knowledge, three histone genes havebeen characterized to date from kinetoplastid protozoa: thegenes coding for histone H2B from Leishmania enriettii(Genske el al., 1991) and for histone H2A from Leish-mania donovani infanturn (Soto et a/., 1992) and T. cruzi(Puerta eta/., 1994). Their transcription products are poly-adenylated. However, it is reported (Genske et a/., 1991)that inhibition of DNA synthesis in L. enriettii does notdecrease the relative level of histone H2B mRNA, suggest-ing that its abundance is not coupled to the Leishmania cellcycle.

We report here the cloning, molecular characterizationand genomic organization of the gene coding for histoneH2B in T. cruzi and show that its transcription product is

polyadenylated. lnhibttlon of DNA synthesis results indecreasing histone H2B mRNA abundance to a very lowlevel, suggesting that histone gene expression is coupledto the cell cycle in Trypanosoma. In accordance with this,while the H2B mRNA is readily detected in the life formsable to divide (epimastigotes in the insect vector andamastigotes in the mammal host), it can be barelydetected in the non-dividing trypomastigotes.

Results

Cloning of a T. cruzi histone HZB cDNA fragment

Taking advantage of the fact that all mRNAs from T. cruzistart with the same 39 nucleotides (the spliced leader,added by trans-splicing to the primary transcription pro-duct of any gene) (De Lange et al., 1984), we cloned the5’ ends of transcripts of interest by synthesizing cDNAusing reverse transcriptase and primers specific for parti-cular sequences, and then amplifying the cDNA bythe polymerase chain reaction (PCR), using the sameprimer and a second one corresponding to the splicedleader sequence. Owing to the low-stringency conditionsin the priming of the reverse transcriptase reaction andbecause in many instances we utilized degenerate firstprimers, it was not unusual to end up with more than oneamplified product (as seen by agarose gel electrophor-esis). By characterizing these bands we have obtaineduseful information on the 5’ ends of numerous T. cruzitranscripts (J. A. Garcia-Salcedo el a/., in preparation). Inan experiment using the oligonucleotide described inthe Experimental procedures, a 200 bp band was ampli-fied, isolated, cloned in an EcoRV-linearized pBSKS-(plasmid p41 EC) and sequenced. The amino-acid-derivedsequence from this cDNA, starting at the first ATG down-stream of the spliced leader, showed strong homology toH2B histones in protein databases. As shown below, thiscDNA fragment does correspond to T. cruzi histone H2B(nucleotides 78-258 plus 30 nucleotides of the splicedleader).

Genomic organization of the T. cruzi histone ti2Bgene

The genomic organization of T. cruzi histone H2B genewas studied by Southern blotting and chromosomal blot-ting of genomic DNA using the cDNA probe. Figure 1Ashows the bands obtained in a blot of DNA digested witha number of restriction enzymes. This pattern concurswith that of a tandemly repeated gene unit of about750 bp. Almost all copies of the gene contain one site forXbal and Sacl. most have two Pstl sites and there is anNcol site that is polymorphic among the repeating copiesand/or alleles. Finally, the enzymes Kpnl and Hindlll do

.

--

1 2 3 4 5 6 B

t .

-- 23

.- VA

-6.4

-- 4A

. w

Fig. 1. Southern blot analysis of the histone Ii28 gene orgamz-atlon. Total DNA trom T cruz~ ep\mastlgotes (1 hrg) was dIgestedwith a number of restnctlon enzymes, electrophoresed. blotted andhybndlzed to digoxlgenin-labelled histone H2B probe (insert fromplasmld p41 EC wlthout spkced leader sequence).A. Lanes l-6 correspond to digestions with Xbel. Sacl, Pstl. Ncol.Kpnl and Hrndlll. respectively.8. Partial digestions with Xbal. Molecular size markers (shown atthe sides in kb) are HindIll digests of lambda DNA.

not cut inside the cluster. To confirm this explanation andto estimate the number of repeating units in the histoneH2B cluster, the experiment shown in Fig. 1 B was carriedout. Partial digestions with Xbal produce bands differing insize by one copy. Since 17 bands can be unambiguouslydetermined from the picture, 18 is probably the minimumnumber of repeats in the cluster.

T. cruzi chromosomes were separated by pulsed-fieldgradient electrophoresis, blotted and hybridized to theH2B probe (Fig. 2, lane 1) and to a probe correspondingto maize histone H3 (lane 2). While the H3 probe hybrid-izes to a chromosome of about 1.7 Mb, the H2B probecomplements sequences in two very large chromosomesof different size. Although the most straightforward infer-ence is that there are two unrelated genomic loci each con-taining a similar number (as judged by the intensity of thebands) of tandemly repeated copies of the histone H2Bgene, it should be borne in mind that T. cruzi is a diploidorganism that often exhibits size heterogeneity betweenallelic chromosomes (Gibson, 1986; Henriksson et al.,1990). On the other hand, the histone H3 locus is notlinked to the H2B loci or to the H2A locus that occurs ina 1 Mb chromosome (Puerta et al., 1994) revealing thatthe genes coding for different histones are not grouped inthe genome of T. cruzi.

Nucleoride sequence and transcription of the H2Bgene

The H2B cDNA probe was used to screen a genomic library

The h/stone H2B gene from Trypanosoma cruzi 1035

of T. crux; DNA in lambda EMBL3 phage (GonzPlez el al.,1990). A posltlve clone was isolated that contalned severalcopies of the H2B gene plus 14 kb of 5’ upstream sequence(data not shown). Copy units were isolated by electrophor-esls after Xbal digestion of phage DNA, purified, subclonedand sequenced. Also, by sequencing upstream of the firstcopy of the cluster in the genomic clone, we could deter-mine the commencement of the repeat. Through directcomparison to the cDNA sequence, it was establishedthat trans-splicing takes place at position 77 of the reportedsequence, behind an acceptor AG; a pyrimidine track,postulated to determine the splice site (Layden and Eisen,1988), appears at position 30 to 53. In the 5’-untranslatedend, the sequence AACCA is repeated three times. Down-stream, the first ATG (position 120) is followed by an openreading frame encompassing 112 codons (teminationcodon TAG at position 456) presenting the usual GC biasof T. cruzi. The amino-acidderived sequence shows thecharactenstics of eukaryotic histone H2B (see the nextsection).

Epimastigote RNA was fractionated on an oligo d(l)-cellulose column, subjected to Northern blotting andhybridized to the cDNA probe. A 670~nucleotide bandappears only in the lane loaded with the polyadenylatedfraction (Fig. 3A); thus, the H2B mRNA is polyadeny-lated, as are histone mRNAs of Leishmania (Genske,1991; Soto et al., 1991), T. cruzi H3 mRNA (our unpub-lished obsen/ation) and H2A mRNA (Puerla et al., 1994).Cytoplasmic RNA from different T. cruzi life stages wassize-separated on formaldehyde-agarose gels, blottedand hybridized to the cDNA probe (Fig. 38). A band ofthe expected size appeared in the lanes loaded withRNA from epimastigotes and amastigotes (i.e. the formsthat are able to multiply), but not with trypomastigote

1 2 34

-2.194

Fig. 2. Pulsed-field gel electrophoresis of T. cruzi chromosomes.Aiter blotting, the filter was hybridized to the histone H2B probe(arrowheads, lane 1) or to a maize histone H3 probe (asterisk, lane2). Hybndizmg chromosomes are marked in an ethidium-bromide-stained lane (lane 3). Also stained, lane 4 displays the chrmsomes of S. cerevrsiae. stram S13, used as markers (the SUBS ofthe two largest chromosomes are shown in kb). For me maize H3probe the hybndization temperature was reduced to 40 C.

.

--

1036 J. A. Garcia-Salcedo. J. L. Oliver. R. P. Stock and A. Gonzdlez

A B

A+ A - T E Av.*‘p

0.3 -

Flg. 3. Northern blot of T. cruzi cyioplasmlc RNA.A. Probmg of poly-A’ (2 pg) and poly-A (10 pg) with an H2B probe.8. Probq of poly-A’ RNA (2 pg) from trypomasltgotes (T), eplmastl-gales (E) and amastlgotes (A).

RNA, indicating that histone H2B RNA accumulates only inthe life forms that are able to divide.

Sl nuclease protection experiments (data not shown)were carried out to map the polyadenylation site ofthe H2B mRNA. The site could accurately be assigned toposition 665 or 666 of the sequence; this was confirmedby isolating a full-size clone from a cDNA library,constructed using amastigote mFlNA (Gonzdlez el al.,1990), and sequencing through the poly-A tail. The H2BmRNA consists of the spliced leader, 42 nucleotidesof 5’-untranslated region, 339 nucleotides of codingsequence, 206 or 207 nucleotides of 3’-untranslated endand a poly-A tail of some 50 residues. Comparison of thecDNA and the genomic sequences results in the following

T.c:

L.e:

discrepancies: G for C (posltion 118) in good agreementwith the polymorphic nature of the Ncol site (Fig. 1A). Afor G (position 126) changes threonlne to alanlne in theamino-acid-denved sequence, G for T (position 243)changes alanlne to senne. It is likely that the cDNA corre-sponds to a gene copy different from the genomic onesequenced.

T. cruzi histone H2B

The H2B gene codes for a 112-amino-acid protein (Fig. 4)with a deduced molecular weight of 12 000 and the usualfeatures of histones H2B: very basic (isolectric pointII .9) and slightly more lysine rich (11.6%) than argininerich (9%). A putative nuclear localization signal (Morelandet al.. 1987) located between amino acids 15 and 23 isplaced just before the transition from hydrophilic to hydro-phobic in the hydropathic plot of the protein. The alignmentof the T. cruziH2B sequence to other histones H2B from anumber of species reveals a 67% identity with L. enrieftii, a47% identity with Saccharomyces cerevisiae, a 46% iden-tity with Caenorhabditis elegans. a 46% identity with seaurchin and a 45% identity with chicken H2B. As previouslyobserved, the amino terminus is the least conserved partamong H2B molecules.

Effect of DNA and protein synthesis inhibitors on H2Bmt?NA abundance

Except for the basal histones, the steady-state level of his-tone mRNAs in higher and lower eukaryotes is regulatedby the cell cycle: histone mRNA concentrations rise byan order of magnitude as the cell enters the S phase, theresult being an increase of both histone gene transcription

RATPKSSSMRKKCCKK3HRKPKRWNVYINR3LKSINNMblASQRLAPRKSSMH........9....V9....A..9.

f-t: APKKAPA.MEKKVKKAPTTL..NKK.RSL.~AI..~KV..QVMPD9.c: SSMEKKPASKAPACKK.MKKTSTSVD... R3KVR.C.Y99..YKV..QTWDT-8: pRKKPMEN~JEKMEKTPACKKPKAEKRLPhCnTA3K~ACC~CKT.CRKKCSI(AK.QVt.YI(I..?)(V..~~PDc-g: P~PAKSAPAPKKGSK.A~KTOK..D..RKK~R.~~Y~~.~..~PDC.e: APPKPSAKO.KKAKKMKP.D...RKMR.tSY9...Y.V..OVHPDD.fl: PPKTSCKAAKKACKAQK.IT.TD.. KK..R.tSYILI..YXV..OVHPD3-p: APTAQVAKKCSKKAVKCTKTAX.... RN..R.LSYO1..YRV..QVHPD

. . . . . . . . . . . . . Y. ..~....M...SI..ruc..L......V.........Pt..............N3CRVCI.KKA.N.~...I..3.....L.SSKL..f..R...9S..V....K.L..R..I3......TK~..STNTCI.~KS.S.L......~.....t..3KLMY...S.~S...I......I..Ct.....VS...R..T)(Y..STOAICI.SKA.S.~...I..I..KL.~.S.KLA.Y...P.IT9..I..9......CC.....VS......TKFT.STGI.SM.C.H......I.... .C..9RLAtW..R3.ITS..I......L..CC.....VS......Tlm.SKTCV.SYJI.S.~......V.....A..SR~..RS.ISS..I......I..C~.....VS......l~.SKTCI.Sr.A.S.n......I.... .A..SRlNfY..RS.lTS..I......L..CE.....VS......TKYT.SK

TCI.SPA.V.fl......I.....C..SRLA~Y...S.1SS..I......~..CE.....VS.... .TlWTTSK

Fig. 4. Ammo acid sequence of hIstone H2B of T. CNZI (T.c.) and homology lo hlstones H2B from L. ennetti (L.e.); Telrahymena fhermophrla(T.1.); S. cerevrsrae (S.C.); Triticum aesnvum (T.a.); Gal/us gal/us (G.g.); C. elegans (Ce.); Drosophrla melanogasfer (Dm.) and Skongylo-cenlrolus purpurafus (Sp.). Dots indxate idenkal amino acids. The EMBL database accession number for the T cruzr histone H2B gene isX60962.

The h/stone H2B gene from Tt-ypanosoma CXUZI 1037

Table 1. Trme course 01 ettecl of aphrdrcokn on H28 mRNA levels,DNA synthess and proletn syntheses.

TimePercentageH28 mRNA

Percentage[3H]-thymrdrnemcorporatron

[35S]-melhronrne(c.p.m.)

0 100 100 40 684.415mrn 73 5 - -3Omrn 65.6 43.2 -45 mm 53.1 - -6Omin - 34.0 -75mrn 90 15.1 -2h 13.0 10.0 65 563.44h 13.5 - -5h6h8h

10h12h14h15h18h20h24h26h28h

13 3130

0.37 -

-26.032.0-47 053.0

169.0186.0205 7

- -9.6 -

- -- 57 475.6

7.8 -- -

4.7 -- -

9.4 -- -

DNA syntheses IS expressed as per cent rncorporabon of [‘t-f]-thymrdrne.Protem synthesis IS expressed as total [35S]-methionrne mcorporationinto TCA-precrprtable material.

and histone mRNA stability (Osley. 1991). Only in onecase. the trypanosomatid L. enn’eltii, has rt been reportedthat the RNA abundance for a non-vanant histone IS notcell-cycle regulated (Genske et al., 1991). We haveextended this study to T. cruzi fit32 mRNA by, in theabsence of a protocol for synchronizing cells. using aphidi-colin, an inhrbitor of DNA polymerase. Incubation of T.cruzi epimastigotes in the presence of 5 rig ml ’ aphidi-colin results in a 90% reduction in DNA synthesis by 2 has measured by [3H]-thymidine incorporation into acid-precipitable material, a level of inhibition maintained up to26 h later (Table 1). Aliquots of the cell culture wereobtained at different times and the relative amount ofH2B RNA was measured by Northern blotting of total cyto-plasmic RNA and hybridization to a t-f26 probe. As acontrol, the blots were also hybridized to a probe corre-sponding to lf6. a tandemly repeated gene coding for aputative calcium-binding protein (Gonzalez el al., 1985).As shown In Fig. 5A, the H2B mRNA abundance declinessteadily for the first 75 min and remains at basal level forabout 4 h but then, surprisingly, increases continuouslywith a doubling time of about 3 h for at least 28 h(Fig. 58, Table 1). The lf8 band remained essentiallyunmodified throughout the time course of the experiment.These results suggest that the H2B mRNA level is

A 6

0 2 4 6 8 12 I4 18 20 24 26 28

25u

I0' -I

0 15 xl 4 5 dl 75 0 2 4 b b IO 12 14 lb lb 20 ?2 24 2b Ib

Fig. 5. Effect of aphrdrcolrn on the relative abundance of H2B mRNA.A A Northern blot of 15 rig total cytoplasmrc RNA from eprmastrgotes cultured in medrum supplemented wrth 5 rig ml - ’ aphidrcolin. Culture ali-quots were taken at 0. 15. 30. 45. 60 and 75 mln of addrtron of aphrdicolrn and hybndrzed with an H2B probe and a 1 f8 probe as control, bothof which were radroactrvety labelled. Below is a graphical representatron of the relatrve abundance of transcripts; the ordinate axis is the per-centage optical densrty of H2B mRNA wrth respect to the 118 control (t =O is 100%) at me indrcated Intervals.8. The same expenment Over a 28h time-course. Culture aliquots were taken at 0. 2. 4. 6. 8. 12. 14. 18, 20, 24, 26 and 28 h (note the recov-ery of mRNA levels In sprte of the ongoing inhibrtron of DNA synthesrs).

.,

. .- - - . ._. _ _ _

1038 J. A. Garcia-Salcedo, J. L. Oliver. R. P. Stock and A. Gonzdlez

controlled by the cell cycle but in a way that is somehowdifferent from that of other eukaryotrc hrstone mRNAs (seethe Dwcuss~on) as the regulation of h&one H2B mRNAabundance seemed to be uncoupled from DNA replica-tron. which was suggested by the fact that mRNA levelskept Increasing even though DNA synthesis was almostcompletely blocked (Fig. 5 and Table 1). It has beenreported that the decline rn H2B mRNA after the additionof aphidicolin is dependent on de nova protein synthesis(Stahl and Gallwrtz, 1977). We find that cycloheximide,an inhibitor of protein synthesis by eukaryotic organisms,impedes the decline in histone H2B mRNA levels(Fig. 6). However, the observed accumulation of H26mFiNA at later times cannot be attributed to the absenceof protein synthesis because [35S]-methionrne incorpora-tion 14 h after the beginning of the expenment is 100%that of the control (Table 1). Furthermore, the inhibition ofprotein synthesis alone is enough to cause a markedincrease in the relative levels of H28 mRNA when com-pared to (l-tubulin mRNA (Fig. 6, lane B.& This ‘super-induction’ of H2B mRNA is completely independent ofthe extent of DNA replication, as the addition of aphidicolindoes not reduce the abundance of the H2B messenger(Fig. 6, lane BIOS) when compared to the control. Table2 shows the quantification of the experiment depicted inFig. 6.

Discussion

Despite an early report to the contrary (Beck and Walker,1964) the presence of histones associated with nuclearDNA has been firmly established in kinetoplastid protozoasuch as C. oncopelti (Leaver and Ramponi. 1971) andT. cruzi (Astolfi 81 al., 1980). Physrcochemrcal character-ization of histone-like proteins suggested that trypano-somatid organisms contain a set of histone proteinssimilar to those found in other eukaryotes. The genes

Flg. 6. Ettect 01 cycloheximide on the abundance of histone H2BmRNA.A. A culture treated wrth 5 trg ml ’ aphrdrcokn at t = 45 min.B. A culture treated with 20 rg ml ’ cyclohexlmlde at I = 0. andSvgml ’ aphtdicolm 45 min later. Cytoplasmlc RNA extracttonswere performed at times indicated (in mrnutes) by the subscnpts.Each lane represents 15 ug cytoplasmrc RNA. As a control, themembrane was hybndrzed to a probe specrlrc for [Mubukn.

Table 2. Effect 01 aphrdrcolm and cyclohexrmrde on protean syntheses,DNA synlhes!s and H2B mRNA abundance.

Protein DNA synthesis H2B mRNATreatment synthesis f%) (%) W)

Control 100 100 100Cyclohexrmrde 24.6 51 .o 200Aphrdrcokn 07.7 12.6 13.0Cyclohexrmtde 27.9 15.6 212.6

+ aphrdrcolrn

DNA synthesis IS expressed as per cent of control cultures. H2BmRNA abundance IS shown relative to [Gtubulrn abundance. Aliquotsof cultures were taken at Ihe times indicated by the subscripts inFig. 6 (mrnutes) and treated as described in the Experirrrenrtll pro-cedures.

coding for histone H2B of I_. enriettii (Genske et a/.,1991) and H2A of L. donovani infanturn (Soto et a / . ,1992) and T. cruzi (Puena et al., 1994) have been clonedand characterized.

We have extended the study of histone genes inTrypanosomatidae by cloning the H2B gene from T.cfuzi. This gene codes for a 112-amino-acid proteinwhose homology to H2B histones decreases from Leish-mania to vertebrates in agreement with the evolutionarydivergence obtained using other methods (Michels andOpperdoes, 1991; Sogin et al., 1986). In contrast to theLeishmania protein, it is as lysine rich as the H2B histonesfrom other eukaryotes and contains a well-conservednuclear localization signal (Moreland et al., 1987) indicat-ing that these signals are conserved in phylogeneticallydistant organisms.

The maximum parsimonious tree obtained (Fig. 7)revealed an extremely divergent nature for the H2Bsequence from 7. cruzi. Bootstrap confidence valueswere obtained not only in the comparison with other Proto-zoa (7. fhermophila) but also with members of the samefamily (L. enrieffi). An extreme divergence has also beenobserved for the histone H4 sequence from 7. cruzi(Toro et al., 1992). Both observations contrast with thewell-known conservatism of histones throughout evolutionand agree with the hypothesis of a clonal origin for theseprotozoa (Tibayrenc et al., 1986).

As much as 51% of 7. cruzj DNA consists of infrequentlyrepeated sequences (Lanar et al., 1981); this fraction isbelieved to be made up of tandemly repeated genes.The H2B gene is repeated at least 18 times in a clusterthat appears on two very large chromosomes of differentmobility. As to whether these chromosomes form an allelicpair of different size or are different chromosomes will bedetermined once additional genetic markers becomeavailable. Allelic chromosomes of different size havebeen shown to exist in the genome of T. cruzi (Gibsonand Miles, 1986; Henriksson ef al., 1990) but, if this isthe case for the H2B-hybridizing chromosomes, their

- 1. c r u z i

- L. enrietti

r T. t he rmoph i l o

S. cerevis iae

!W T. aest ivum

G. gallus

C. e legans

4 D. melanogoster4.

M. g l a c i o l i s

m 7. Unrooted phylogenetlc tree based on sequences of the H2B&m. obtalned by maximum parsimony. Numbers al the nodes of-her represent bootstrap confidence values obtamed from 100~tlons.

dffference in size would be surprisingly large. On the otherhnd, both an H2A probe and a maize H3 probe hybridizek a different T. cruzi chromosome, indicating that no link-age Is found among different histone genes in 7. cruzi, asoccurs in other eukaryotes.

Tandemly repeated genes are transcribed polycistroni-caky in trypanosomatids (Gonzalez ef al., 1985; Muhichand Boothroyd, 1988; Murphy et a/., 1986). Pre-mRNAsare processed by rranssplicing and polyadenylation toproduce translatable RNAs and intergenic sequencesthat are degraded. It is not surprising, then, that the H2BmRNA is polyadenylated, as polyadenylation is probablynecessary for the processing of pre-mRNA transcribedfrom tandemly repeated genes. We have been able todetermine the beginning of the H2B repetitive cluster andhave found that trans-splicing of the H2B pre-mRNAtakes place at the first AG in the repeated sequence. Thesignificance of this, if any, is unknown at the moment,because, to the best of our knowledge, the beginning ofany other cluster, which could be used for comparative pur-poses, is still to be characterized.

Inhibition of 7. cruziDNA synthesis by aphidicolin resultsin the reduction of H2B mRNA abundance to a basal level,suggesting that histone gene expression is coupled to thecell cycle, as is the case in yeast and higher eukaryotes.Accordingly, no H2B mRNA can be detected in the trypo-mastigote stage of the parasite, which is unable to multi-ply. However, a progressive recovery in H2B mRNAabundance can be observed with time that cannot beimputed to the concomitant decrease in protein synthesisneeded for the mRNA level decrease, nor to a reinitiationof DNA synthesis. Genske et al. (1991) after studyingthe histone H2B expression in L. enrieflii, concludedthat it is unrelated to the cell cycle because the addition of

The h/stone H2B gene tram Trypanosoma cruzl 1039

aphidicolin did not result In a reduction in H2B mRNAabundance. Although it cannot be ruled out that leish-manlal H2B expressron IS regulated differently from itstrypanosomal counterpart, our results might explain theapparent disagreement. The frrst time point reported inthat study is taken 8 h after the addition of aphidicolin. Bycomparison to the curve In Fig. 5B. and bearing in mindthe somehow faster metabolism and cell cycle of Leish-mania, 8 h may be sufficient for recovery of H2B mRNAlevels.

The regulation of mRNA abundance involves transcrip-tional as well as post-transcnptional mechanisms, thelatter acting both at the level of processing and at thelevel of messenger stability. Peltz and Ross (1987) haveshown that the presence of free histones accelerates thedegradation of their mRNAs in a cell-free system. Theyhave argued that the accumulation of free histones in acell in which DNA synthesis has been inhibited would be(at least partially) responsible for the concomitantdecrease in their respectrve mRNAs. This would takeplace by a hypothetical interaction between the histoneand its messenger which would render the mRNA moresusceptible to nuclease attack. Interestingly, the 3’ end ofyeast H2B mRNA has been reported to confer cell-cycleregulation of its abundance by specific destabilization ofreporter gene constructs (Xu et al., 1990).

Formally, the hypothesis that transcriptional control ofH2B expression is at work cannot be ruled out. Never-theless, we feel inclined to speculate that H2B mRNAlevels are regulated post-transcriptionally, in an auto-genous mechanism in which histone H2B usage is a keyregulatory element. The reasons for this belief are: first, ifinhibition of DNA replication is taken as the sole stimulusnecessary to stop transcription of the H2B genes, it ishard to explain why it is later reinitiated, whilst DNA repli-cation (the original down-regulatory stimulus) remainsblocked; second, a replication-coupled transcriptionalcontrol of H2B mRNA levels would not explain the super-induction of H2B mRNA observed when de novo proteinsynthesis is inhibited, as DNA replication inhibition cannotovercome the up-regulatory effect of protein synthesis inhi-bition (Fig. 6 and Table 2). The hypothesis that histoneH2B is at least one of the regulatory agents of H2BmRNA abundance is suggested by the work of Peltz andRoss (1987), and also by the fact that this would explainthe drop and subsequent increase in H2B levels - adecrease after histone requirements are diminished byDNA synthesis inhibitlon and a recovery after depletionof the cytoplasmic pool of the protein, a depletion whichalso must occur (and more rapidly) when protein synthesisis blocked with ongoing DNA replication, thus explainingthe rapid ‘superinduction’ observed.

Both Sl nuclease protection studies (data not shown)and computer predictions have revealed a very complex

1040 J. A. Garcia-Salcedo. J. L. Oliver, R. P. Stock and A. Gonztilez

secondary structure for the 3’ end of the H2B mRNA, whichcould provide a framework for InteractIons of the type pro-posed. The relevance of this fInding IS currently understudy rn our laboratory.

Experimental procedures

Growth of parasites and isolation of nucleic acids

The Y strain of T. cruz~ (Silva and Nussenswelg, 1953) wasused throughout this work. Epimastigotes were grown Inliver Infusion tryptose (LIT) medium supplemented with 10%heat-mactwated fetal calf serum at 26 C with gentle shaking(Castellani et a/., 1967). Trypomastigotes were collectedfrom the supernatant of infected monolayers of LLC-MK2eplthelial cells (Andrews and Colli, 1982). Extracellularamastlgotes were obtained by incubating recently releasedtrypomastlgotes in LIT medium at 37 C under 5% CO1 for24 h (Andrews et al.. 1987).

Cells were lysed with 1% NP40 in the presence of 10 mMvanadyl complexes (Sambrook et a/., 1989). After centrifuga-tlon. DNA was purified from the nuclear pellet using theSDS-Protelnase K method (Gross-Bellard el a/., 1973); cyto-plasmlc RNA was obtained from the supematant as reported(Gonzalez el al., 1990). The polyadenylated fraction ofthe RNA was obtained by chromatography on an oligo(dT)-cellulose column (Aviv and Leder, 1972).

Southern, Northern and chromosomal blot analysis

T. CNZI DNA was digested with restriction enzymes andelectrophoresed in agarose gels using TAE buffer containingethidium bromide, depurinated in 0.25M HCI for 15min andtransferred to nylon membranes in 0.5M NaOH (Sambrooket a/., 1989). For molecular karyotype analysis (Schwartzand Cantor, 1984), approximately 8 x 10’ epimastigotes perplug of low-melting-point agarose were treated with SDSand Proteinase K (Van der Ploeg et al., 1984). Pulsed-fieldgel electrophoresis was performed in an LKB 2015 PulsphorSystem apparatus using 1% agarose gels and TBE buffermaintained at 12°C. The running conditions were 350, 500,750 and 1000 second pulse times for 24 h each at 84 V.Chromosomes were visualized by incubation with ethidiumbromide, depurinated and transferred to nylon as above.RNA was denatured and size-separated by electrophoresisin 1% agarose gels containing formaldehyde in MOPS buf-fer, pti 7.0. Transfer to nylon membranes was carried out in50 mM NaOH by capillarity.

Membranes were neutralized by washing with 2 x SSC andthen incubated at 42°C for at least 3 h in prehybrfdization solu-tion consisting of 50% formamide, 5 x SSC, 50mM sodiumphosphate, pH 6.8, 5 x Denhardt’s (50 x Denhardt’s is 1%each of Ficoll 400, polyvinylpyrrolidone and bovine serumalbumin) and 0.2% SDS. Probes were labelled using [z-~~P]-dCTP by the random primer method (Feinberg and Volgel-stein, 1983). To the prehybridization solution was addeddenatured probe (2 x 1O”c.p.m. ml-’ of hybridization solu-tion) and salmon sperm DNA (0.2 mg ml-’ final concentra-tion). Hybridization was carried out overnight at 42 C. After afinal wash at 55 C in 0.1 x SSC and 0.1% SDS, the filters

were exposed to XAR5 films (Kodak). Densltometric analyseswere performed by scanning the autoradiograms with 2BioRad 620 densitometer and the peak areas were automatl-tally integrated using the BioRad 1 -D Analyst software. WhenIndicated, the probe was labelled with digoxigenin-dUTP(Eoehnnger Mannhelm) and hybridizations were carried OUI.as suggested by the manufacturer, at 68 C.

Library screening, subclonrng and sequence analysis

Standard procedures (Sambrook et al.. 1989) were used toscreen with radioactive probes and to isolate positive clonesfrom an amastigote cDNA library constructed in lambdagtl 1, and a T. cruzl genomic library in lambda EMBLB (Gon-z6lez et a/., 1990). Restriction or PCR-amplified fragmentswere subcloned into Bluescnpt (Stratagene) or Geminis (Pro-mega) plasmids, used to transform Escherrchia co/iXL-1 Blue.For all purposes, plasmids were purified by the alkaline-SDSminiprep method (Birnboim and Daly, 1979) scaled up for15 ml culture volume. Sequencing was performed on bothstrands by Sanger’s dideoxy protocol (Sanger et al., 1977)using Klenow or phage T7 DNA polymerases. Sequence ana-lysis was carried out using the University of Wisconsin GCGsoftware package (Devereux et al., 1984).

Primer extension and PCR amplification

cDNA was synthesized from epimastigote poly-A’ RNA byAMV reverse transcriptase using as primer the oligonucleo-tide 5’-GGNAYNCKNACYTCNCC-3’. The RNA templatewas hydrolysed with NaOH and the cDNA passed through aSephadex G-50 spun minicolumn. PCR amplification wascarried out using the same primer and a second one, 5’-CGCTAlTAlTGATACAGllTCTG-3’, in a thermocycler setat 1.5min x 94 C, 1.5 min x 48 C and 3 min x 72°C for 30cycles. Reaction products were separated by electrophoresisin agarose gels and the DNA from gel slices containing visiblebands were isolated using GeneClean (Bio 101).

Measurement of the rate of DNA and proteinsynthesis

One-hundred microlitres of culture samples of 35 x 10’epimastlgotes per ml in DMEM plus 10% heat-inactivatedfetal calf serum were pulsed for 30 min with 30 PCi ml - ’ of[3H]-thymidine or 10 PCi ml - ’ [?+methionine. The pulsewas terminated by the addition of 1 ml of cold 10% trichloro-acetic acid (TCA). The precipitate was filtered through glassmicrofibre filters and washed twice with 5ml cold 5% TCA.The filters were dried at 60’ C for 30 min and bound radioactiv-ity was counted in 4 ml of scintillation liquid.

Phylogenetic tree construction

The H2B sequence from T. cruzi was compared to eighthomologous sequences retrieved from the Swiss-Prot proteinsequence database. The following taxa and accessionnumbers were used: L. enrietfi accession number P27893;Terrahymena thermophila accession number P08993; yeast

(S. cerevrsae) accession number PO2293 wheat (Trifrcum~esffvum) acccsslon n u m b e r P27807; sptny starfrsh(Marthastenas glacralfs) accession number PO2285 nema-tode (C. elegans) accesston number P04255; fruit fly (Droso-phi/a melanogaster) accession number P02283; and chicken(Gel/us gal/us) accession number P02279. Sequences werealigned using the overall multiple alignment algonthm(Higgtns and Sharp, 1989); statistical confidence in thetopologies was assessed using the bootstrap algonthm with100 repkcations. To this end, the aligned sequences weresubfected to analysis by several programs from the PHYLIP

(Version 3.5~~ March 1993) phylogeny inference package(Felsenstein, 1988). First, we used SECIEOOT to produce 100bootstrapped data sets. Then, we ran PAOTPARS to obtainthe maxlmum parsimony trees for each one of thesedata sets. Lastly, the program CONSENSE allowed us to obtatnthe majority rule consensus tree with bootstrap confidencevalues.

Acknowledgements

We thank Dr Enric Espel (Centro de Investigacidn y Desar-rollo, CSIC. Barcelona) for his kind gift of the maize H3probe. This work was supported by Grant PB89-0064 fromthe Direcctdn General de Investigation Cientifica y Tecnica(Spain). J.A.G.S. is a recipient of a fellowship from the Span-ish Ministerio de Education y Ciencia.

References

Ajiro, K., and Nishimoto, T. (1985) Specific site of histone H3phosphorylation related to the maintenance of prematurechromosome condensation. J Biol Chem 260: 15379-15381.

Andrews, N.W., and Colli, W. (1982) Adhesion and inter-iorization of Trypanosoma cruzi in mammalian cells. JProfozool29: 264-269.

Andrew% N.W., Hong, K., Ftobbins, E.S., and Nussenzweig,V. (1987) Stage-specific surface antigens expressed duringmorphogenesis of vertebrate forms of Trypanosoma cruzi.Exp Parasrfol64: 474-484.

Astolfi, S.. Martin de Sa, C., and Gander, E.S. (1980) On thechromatin structure of Tfypanosoma cruzi. MO/ B&hemParasifol 1: 45-53.

Aviv, H., and Leder, P. (1972) Purification of biologicallyactive globin messenger RNA by chromatography onoligothymidylic acid-cellulose. Proc Nat/ Acad Sci USA69: 1408-1411.

Baumbach, L.L.. Stein, G.S., and Stein, J. (1987) Regulationof human histone gene expression: transcrfptional andposttranscriptional control in the coupling of histonemessenger RNA stability with DNA replication. Bioohem-isfry 26: 6178-6187.

Beck, J.S., and Walker, P.J. (1964) Antigenicity of Trypano-soma nuclei: evidence that DNA is not coupled to histone inthese protozoa. Nature 204: 194-l 95.

Bender, K., Betschart, B., Shaller, J., Kampfer, U., andHecker. H. (1992) Biochemical properties of histone-likeproteins of procyclic Trypanosoma brucei brucei. AcfaTropica 50: 169- 184.

The hisfone HZfI gene from Trypanosoma cruzi 1041

Bimboim. H.C.. and Doly, J. (1979) A rapid alkaline extractionprocedure for screentng recombinant plasmtd DNA. NuclAcid Res 7: 1513-1523.

Bimstiel, M.L.. Bussltnger, M.. and Strub. K. (1985) Tran-scription terminatton and 3’ processing: the end is in site.Cell 41: 349-359.

Busch, H., and Goldknopf, I.L. (1981) Ubiquitin-proteinconjugates. Mol Cell B&hem 40: 173- 187.

Castellani, O., Ribeiro, L.V.. and Femandes, J.F. (1967)Differentiation of Trypanosoma cruel in culture. J Profozool14: 447-451.

De Lange, R.J., Hooper, J.A.. and Smith, E.L. (1973) HistoneIll. J Biol Chem 248: 3261-3274.

De Lange, T.. Berkvens, T.M., Veerman, H.J.G., Frasch,A.C.C., Barry, J.D., and Borst. P. (1984) Nucl Acid Res 12:4431-4443.

De Souza. W. (1984) Cell biology of Trypanosoma cruzi. lnfRev Cflo/ 66: 197-283.

Devereux, J., Haebem, P.. and Smithies, 0. (1984) Acomprehensive set of sequence analysis programs forVAX. Nucl Acid Res 6: 5725-5737.

Doenecke, D., and Gallwitz, J.J. (1982) Acetylation ofhistones in nucleosomes. Mel Cell B&hem 44: 113-l 28.

Duschak, V.G., and Cazzulo. J.J. (1990) The histones of theinsect trypanosoatid Ctifhidia fasciculafa. Biochim BiophysAcfa 1040: 159- 166.

Elpidina, E.N., Zaitseva, G.N., and Krasheninnikov, J.A.(1979) Histones from Trypanosoma lewisi nuclei. Bio-khimiya 44: 1830-1641.

Feinberg, A.P.. and Volgelstein, 8. (1983) A technique forradiolabelling DNA restriction fragments to high specificacitivity. Anal B&hem 132: 6-l 3.

Felsenstein, J. (1988) Phylogenies from molecularsequences: inference and reliability. Annu Rev Genetics22: 521-565.

Genske. J.E., Cairns, B.R., Stack, S.P., and Landfear, SM.(1991) Structure and regulation of histone H2B mRNAsfrom Leishmania enrieffii. MO/ Cell Biol 11: 240-249.

Gibson, WC., and Miles, M.A. (1986) The katyotype andploidy of Trypanosoma cruzi. EMBO J 5: 1299-l 305.

Gonzalez, A., Lemer, T.J., Huecas, M., Sosa-Pineda, B.,Nogueira. N.. and Lizardi, P.M. (1985) Apparent generationof a segmented mRNA from two separated tandem genefamilies in Trypanosoma cruzi. Nucl Acid Res 16: 5789-5804.

Gonzalez, A., Rosales, J.L., Ley, V., and Diaz, C. (1990)Cloning and characterization of gene coding for a protein(KAP) associated with the kinetoplast of epimastigotes andamastigotes of Trypanosoma cruzi. MO/ B&hem Parasifol40: 233-244.

Gross-Bellard, M., Oudet, P.. and Chambon, P. (1973)Isolation of high-molecular-weight DNA from mammaliancells. Eur J B&hem 36: 32-38.

Harris, M.E., BBhni, R., Schneiderman, M.H., Ramamurthy,L., Schijmperfi, D., and Marzluff, W.F. (1991) Regulation ofhistone mRNA in the unperturbed cell cycle: evidencesuggesting control at two posttranscriptional steps. MO/ CellBiol 11: 2416-2424.

Heintz. N., Sive, H.L.. and Roeder, G. (1983) Regulation ofhuman histone gene expression: kinetics of accumulationand changes In the rate of histone gene transcription and

..

1042 J. A. Garcia-Salcedo, J. l_. Oliver, R. P. Stock and A. Gonzalez

histone mRNA levels In mouse myeloma cells. MO/ Cell B/o/3: 539-550.

Hennksson. .I., Aslund. L., Maana. R.A.. Franke de Cazzulo.B.M.. Cazzulo. J.J., Frasch, A.C.C., and Pettersson, U.(1990) Chromosomal localrzatron of seven cloned antigengenes provrdes evidence of diploidy and further demon-stration of karyotype variability in Trypanosoma cruz~ MO/Biochem Paraslfol42: 213-224.

Higgins, D.G.. and Sharp, P.M. (1989) Fast and sensitivemultiple sequence alignments on a microcomputer. CompAppl Bioscr (CAB/OS) 5: 11 !!I- 119.

Lanar. D.E.. Silven Levi, L., and Manning, J.E. (1981)CornposItron of T. cruzi DNA. M 8 B 3: 327-341.

Layden, R.E., and Eisen, H. (1988) Alternate trans-splicing InTrypanosoma equiperdum : implications for splice sateselection. MO/ Cell Biol8: 1352- 1360.

Leaver, J.L., and Ramponi, G. (1971) Occurrence of histonesin the trypanosomatid flagellate Cnfhidia oncopelfi. Bio-them J 125: 44.

Levy-Wilsson. 8. (1983) Glycosylation. ADP-ribosolation andmethylatron of Tetrahymena histones. BiochemWry 22:484-409.

Lewin, B. (1980) Chromatin components: histones. In GeneExpressron 2. New York: John Wiley & Sons, pp. 299-321.

Lycan, D.E.. Osley, M.A., and Hereford, L.M. (1987) Role oftranscriptional and posttranscriptional regulation in expres-sion of histone genes in Saccharomyces cerevisiae. MolCell Biol7: 614-621.

Mannironi. C.. Bonner, W.M., and Hatch, CL. (1989) H2A.X.a histone rsoprotein with a conserved Cterminal sequence,is encoded by a novel mRNA with both DNA replicationtype and poly-A 3’ processing signals. Nucl Acid Res 17:9113-9126.

Marashi. F.. Baumbach, L., Rickles. R., Sierra, F., Stein, J.L..and Stein, G.A. (1982) Histone proteins in HeLa S3 cellsare synthesized in a cell-cycle stage specific manner.Science 215: 683-685.

Maxson, R., Cohn, R., and Kedes, L. (1983) Expression andorganization of histone genes. Annu Rev Genet 17: 239-277.

Michels, P.A.M., and Opperdoes, F.R. (1991) The evolutionaryorigin of glycosomes. Parasitology Today 7: 10% 1 OS.

Moreland, R.B.. Langevin, G.L., Singer, R.H., Garcea, R.L.,and Hereford, L.M. (1987) Amino acid sequences thatdetermine the nuclear IoCalization of yeast histone 28. MO/Cell Biol7: 4048-4057.

Muhich, M.J., and Boothroyd, J.C. (1988) Polycistronictranscripts in trypanosomes and their accumulation duringheat shock: evidence for a precursor role in mRNAsynthesis. Mol Cell Biol8: 3837-3848.

Murphy, W.J., Watkins, J.K., and Agabian, N. (1986)Identification of a novel Y branch structure as anintermediate in Trypanosoma mRNA processing: evidencefor trans splicing. Cell 47: 517-525.

Osley, MA. (1991) The regulation of histone synthesis in thecell cycle. Annu Rev Biochem 66: 827-861.

Pandey, N.B., and Marzluff. W.F. (1987) The stem-loopstructure at the 3’ end of histone mRNA is necessary andsufficient for regulation of histone mRNA stability. MO/ CellBiol7: 4557-4559.

Pelt& S., and Ross, J. (1987) Autogenous regulation of

histone mRNA decay by histone proteins In a cell-freesystem. Mol Cell &o/7: 4345-4356.

Puerta. C., Martin, J.. Alonso. C., and Lopez, M.C. (1994)Isolation and characterization of the gene encoding histoneH2A from Trypanosoma cruzi. MO/ Biochem Parasltol 64:l-10.

Rubio, J., Rosado. Y.J., and Castarieda, M. (1980) Subunitstructure of Trypanosoma cruz/ chromatrn. Can J Biochem58: 1247-1251.

Sambrook, J., Fritsch. E.F., and Manratis, T. (1989) MolecularClonmg: A Laboratory Manual. 2nd edn. Cold SpringHarbor, New York: Cold Spnng Harbor Laboratory Press.

Sanger, F., Nicklen, S., and Coulson. A.R. (1977) DNAsequencing with chain-terminating inhibitors. Proc Nat/Acad Sci USA 74: 5463-5467.

Silva. L.H.P., and Nussensweig, V. (1953) Sobre uma cepade Trypanosoma cruzi altamenta virulenta para o camun-dongo branco. Fofia C/in Biol20: 197-203.

Simpson, L. (1987) The mitochondrial genome of kinetoplas-tid protozoa: genomic organization, transcription, replica-tion and evolution. Annu Rev Microbial 41: 363-382.

Simpson, R.T. (1978) Structure of chromatin containingextensively acetylated H3 and H4. Cell 13: 691-699.

Sittman, D.B., Graves, R.A., and Marzluff, W.F. (1983)Histone mRNA concentrations are regulated at the levelof transcription and mRNA degradation. Proc Nat/ Acad SciUSA 80: 1849-1853.

Sogin, M.L., Elwood, H.J., and Gunderson, H.H. (1986)Evolutionary diversity of eukaryotic small-subunit rRNAgenes. Proc Nat/ Acad Sci USA 83: 1383-l 387.

Solari, A.J., Tandler. C.J., and Duschak. V.G. (1985) Finestructure and cytochemistry of the mitotic plaques ofrrypanosoma cruzi and Crifhidia fasciculafa. J SubmicroscCyioll7: 583-591.

Soto. M., Requena, J.M., Jimenez-Ruiz, A., and Alonso, C.(1991) The mRNA coding for the nucleosomal protein H2Aof Leishmania is polyadenylated and has stem-loops at the3’ end. Nucl Acid Res 19: 4554.

Soto, M., Requena, J.M., Gomez, L.C., Navarrete, I., andAlonso, C. (1992) Molecular characterization of a Leish-mania donovani infantum antigen identified as histoneH2A. Eur J Biochem 205: 211-216.

Stahl, H.. and Gallwitz, D. (1977) Fate of histone messengerRNA in synchronized HeLa cells in the absence of initiationof protein synthesis. Eur J Biochem 72: 385-392.

Stauber, C., and Schumperti, D. (1988) 3’ processing of pre-mRNA plays a major role in proliferationdependentregulation of histone gene expression. Nucl Acid Res 16:9399-9414.

Schwartz, DC., and Cantor, CR. (1984) Separation of yeastchromosome-sized DNA by pulsed field gradient gelelectrophoresis. Cell 37: 67-75.

Tibayrenc, M., Ward, P., Moya, A., and Ayala, F.J. (1986)Natural populations of Trypanosoma cruzi, the agent ofChagas’ disease, have a complex multiclonal structure.Proc Nat/ Acad Sci USA 83: 115-119.

Toro. G.C., and Galanti. N. (1990) Trypanosoma cmzihistones. Further characterization and comparison withhigher eukaryotes. Biochem tnt Exp 21: 481-490.

Toro, G.C., Wemstedt, C., Medina. C., Jaramillo, N., Hellman.U., and Galanti, N. (1992) Extremely divergent histone H4

sequence from Tqpanosoma cruzt: evoluttonary implica-trans. J Cell Biochem 49: 266-271.

Van der Ploeg, L.H.T.. Schwartz, D.C.. Cantor, C.R., andBorst. P. (1984) Antigenic vanation in Trypanosoma bruceianalysed by electrophoresis separation of chromosome-sized DNA molecules. Cell 37: 77-84.

Wells, D.. and Kedes, L. (1986) Structure of a human histonecDNA: evidence that basally expressed histone geneshave interventng sequences and encode polyadenylatedmRNAs. Proc Nat/ Acad Scr USA 82: 2834-2838.

World Health Organization (1989) Troprcal Diseases,

The h/stone HZ8 gene from Trypanosoma cruzi 1043

Progress m lnfematronal Research. 1987- 1988. 9thProgramme Report. UNDP/Wond BankMI.H.0. SpecialProgramme on Research and Training in Tropical Dis-eases.

Wu. R.S.. and Bonner, W.M. (1981) Separation of basalhistone synthesis from S phase histone synthesis individing cells. Cell 27: 321-330.

Xu, H., Johnson, L., and Grunstein, M. (1990) Coding andnoncoding sequences at the 3’ end of yeast histone H2BmRNA confer cell cycle regulation. MO/ Cell BiollO: 2687-2694.