Eur. J. Biochem. 205,211 -216 (1992) Q FEBS 1992

Molecular characterization of a Leishmania donovani infanturn antigen identified as histone H2A Manuel SOTO', JOSC Maria REQUENA', Luis Carlos GOMEZ', Ignacio NAVARRETE2 and Carlos ALONSO'

' Departamento de Medicina y Sanidad Animal, Facultad de Veterinaria, Universidad de Extremadura, CBceres, Spain

(Received September 23/December 3, 1991) - EJB 91 1263

Centro de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Spain

A Leishmania donovani infantum promastigote cDNA expression library was screened with a serum obtained from a dog naturally infected with this parasite. One of the positive clones obtained revealed nucleotide sequence similarities with the histone H2A genes from various organisms. North- ern blot analyses and sequence data of three independently isolated cDNA clones indicated that the Leishmania H2A mRNAs are polyadenylated, as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast. The analysis of the genomic distribution of the DNA coding for histone H2A suggested that, in L. d. infuntum, there are at least four genes coding for the H2A protein. It is likely that there is a simultaneous expression of at least two of the H2A genes since differences in nucleotide sequence between two of the sequenced cDNAs were observed. Affinity- purified antibodies against the P-galactosidase-fused H2A protein recognize specifically a Leishmania protein band with a molecular mass of 14 kDa.

Leishmania are protozoan parasites of vertebrates which have two discrete life stages. They exist as extracellular pro- mastigotes within the gut of the insect vector and as intracellu- lar amastigotes in mononuclear phagocytic cells of the host, such as macrophages, after having been introduced into the mammalian host by the insect vector. The life cycle is com- pleted when a sandfly bites an infected host and takes a blood meal, ingesting the parasite-laden macrophages [l]. Although it seems that the humoral response against Leishmania in- vasion is not the most important effector mechanism for the immune resolution of leishmaniasis, it is known that the host immune system ellicits high levels of circulating specific anti- bodies against Leishmania antigens [I]. During infection with Leishmania [2] and other pathogens [3], however, a high per- centage of the antibodies which arise in the host are directed against conserved proteins, among which the members of the 70-kDa heat-shock protein (hsp70) family have been found to be the major targets of the host immune system. Similarly, high levels of antibodies directed against actin and tubulin were found in the sera of dogs chronically infected with L. donovuni [4]. Moreover, proteins associated with DNA, chromatin and RNA (including histones, DNA topoisomer- ases and small ribonucleoproteins) have been identified as targets of the autoimmune response (for review see [5]).

In the present paper we describe the genomic organization and expression of L. d. infantum H2A genes and show that,

Correspondence to C. Alonso, Centro dc Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Canto Blanco, E-28049 Madrid, Spain

Ahhreviutions. ORF, open rcading frame; UTR, untranslated region; hsp70, 70-kDa heat-shock protcin.

Note. The nucleotide scquence data have becn deposited in the EMBL sequence data bank under thc accession numbers X60054 and X61936.

in the sera of L. d. injantum-infected dogs, there are antibodies against histone H2A. This protein, like the entire histone family, is probably one of the most conserved proteins across the phylogenetic spectrum [6], reflecting its apparent univer- sality of function relative to cell division and DNA packing. Although the studies concerning chromatin structure and composition of trypanosomatids are scanty, the existence of nucleosomes has been described in Trypanosoma cruzi [7] which include a set of core histones similar, but not identical, to those present in higher eukaryotes [8]. Moreover, based on their electrophoretic mobility, the existence of histones hdS also been described in Crithidiu fasciculata [9]. However, to date, the only histone gene that has been isolated and studied from members of the order Kinetoplastida is the histone H2B gene of L. enriettii [lo]. We present data showing that several genes coding for the H2A protein must exist in the genome of L. d. infantum and that at least two of them are simultaneously expressed during the promastigote stage of the parasite. This is also the first report of a n immune response against the H2A histone protein during infection with parasitic pathogens.

MATERIALS AND METHODS Growth of parasites

Promastigotes of L. d. infantum (LEM75; zymodeme 1) were grown at 2 6 T in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Flow Lab., Irvine, UK).

Sera Sera were obtained from dogs affected with visceral leish-

maniasis from the Mediterranean and Extremadura areas of Spain.

21 2

Construction and screening of cDNA libraries

A igt l 1 expression library of lo6 recombinant phages was constructed from L. d. infanturn promastigote poly(A)-rich RNA using the manufacturer’s protocols (Pharmacia). The phages were plated on an Escherichia coli Y1090(r-) strain (Stratagene) and screened with a Leishmania-infected dog serum (called SL9) as described [ll]. Radioiodinated protein A (Amersham) was used as the secondary screening reagent. Three different recombinant phages called cL7, cL71 and cL72 were isolated for analysis.

Subcloning of the recombinant phages and expression of the fusion protein

Onc of the selected positive recombinant Lgtll phages, called cL7, was digested with EcoRI and the insert was sub- cloned in the EcoRl site of the pMS plasmid. This modified pUR expression vector was constructed in our laboratory by delction of the EcoRI site located downstream of the polylinker site of pUR291 [12]. For our purposes, the pMS plasmid has two advantages: (a) it permits the direct cloning of the insert derived from a recombinant Agtll phage in the single EcoRI-cut site of pMS; (b) the colonies carrying the recombinant plasmids are colourless. The resulting rc- coinbinant plasmid was then called pMS-cL7. The induction of expression of the /l-galactosidase-fused protein was carried out as described [13]. The cell proteins were separated by preparative gel electrophoresis [I41 after suspension of the cells in 1/15 vol. of a lysis buffer (50 mM Tris/HCI pH 6.8, 100 mM 2-mercaptoethanol, 2% SDS, 10% glycerol, 0.05% bromophenol blue).

Southern and Northern blot analysis

The L. d. i~~fiznturn DNA and RNA were isolated as pre- viously described [15, 161. To study the genomic distribution of the insert of the isolated cL7 clone, the parasite DNA was digested with a variety of restriction enzymes, subjected to electrophoresis in 0.8% agarose gels and transferred to nylon membranes (Hybond-N, Amersham). To study the expression of the insert of this clone, total RNA was size-separated on agarose/formaldehyde gels as described [17] and transferred to nylon membranes. Hybridizations, either for DNA or RNA analysis, were performed in 50% formamide, 6 x NaCl/Cit (1 x NaCl/Cit = 15 mM sodium citrate, 150 mM sodium chloride, pH 7.0), 0.1 YO SDS and 0.25 mg/ml of herring sperm DNA at 42°C overnight. Final post-hybridization washes were performed in 0.1 x NaCl/Cit and 0.2% SDS at 50°C for 1 h.

DNA sequencing

The DNA inscrts of clones cL7, cL71 and cL72 were subcloned into the EcoKl site of both M13mp19 and pUC8. DNA sequencing was performed by the enzymatic method of Sanger et al. [18] using the ‘sequenase’ enzyme (United States Biochemical Corp.). Also, two internal oligonucleotides were used as primers Tor subsequent sequencing reactions. The nucleotide sequence of both strands was determined. The analysis of the DNA and amino acid sequence was done using the University of Wisconsin Genetics Computer Group programs “191 and by accessing to the GenBank and EMBL data bases of protein and DNA sequences (September 1990).

Affinity purification of antibodies against the P-galactosidase-fused protein and Western blotting

Specific antibodies against the pMS-cL7 expressed fusion protein were affinity-purified from the SL9 serum on an anti- gen column. For that purpose, total proteins from the induced cells carrying the pMS-cL7 plasmid were separated by SDS; PAGE and the fusion protein was electroeluted from the gel. The eluted protein was dialyzed against coupling buffer (0.1 M NaHC03, 0.5 M NaCl), covalently bound to CNBr-activated Sepharose 4B (Pharmacia) and mounted into a column. The specific antibodies were eluted from the column with 0.1 M glycine pH 2.8. Afterwards, the buffer was re-equilibrated to pH 7.5 with 1 M Tris/HCl.

Protein samples were prepared from I O9 Leishmania pro- mastigotes. The cultured parasites were washed three times with Tris-buffered saline (10 mM Tris/HCl pH 8.0, 130 mM NaCl, 5 mM KC1, 1 mM EGTA), incubated in ice for 15 min in 0.2 ml of a lysis buffer containing 10 mM Tris/HCl pH 8.0, 1% Triton X-100, 0.15 M NaCl, 1 mM phenylmcthylsulfonyl fluoridc and sonicated for 10 min in a water bath. Samples of 5 p1 (approx. 20 pg total proteins) were mixed 1: 1 with 2 x SDSjPAGE buffer [I41 and separated in 12% polyacryl- amide gels using the Mini-protean system (BioRad). After electrophoresis, the proteins were transferred to nitrocellulose membranes (Amersham). A peroxidase immuno-conjugate (Nordic Immunology) was used as second antibody (dilution 1 : 2000). The specific binding was revealed with the western blotting detection ECL system (Amersham).

RESULTS

Identification of histone H2A cDNA clones

After screening of a cDNA library with a visceral leish- maniasis serum (SL9) a recombinant Lgtl1 clone, called cL7, was isolated having a 516-bp cDNA insert. A rapid database comparison of the sequence of this insert revealed nucleotide similarities with the histone H2A gene sequence of other or- ganisms. Since the insert of clone cL7 lacks the nucleotide sequence coding for the N-terminal amino acids of the known histone H2As, it was necessary to obtain a clone containing a full-length cDNA. For that purpose the cDNA library was screened using as probe the insert of cL7. Two cDNAs clones, called cL71 and cL72, containing inserts larger than that of cL7 were finally chosen for analysis. The nucleotide sequences of the inserts of clones cL71 and cL72 are shown in Fig. 1. No differences in nucleotide sequence exist between the inserts of cL7 and cL71, except that the cL7 insert was 173 bp shorter than the insert of cL71. However, it may be observed that the nucleotide sequence of the insert of cL72 is not identical to the nucleotide sequence of the insert of cL71 (Fig. 1; see below).

Two open reading frames (ORF) of similar length are present in the nucleotide sequence of the insert of clone cL71. ORFl is open at the second nucleotide of the sequence and ends at nucleotide 493.ORF2 starts at nucleotide 81 and ends at nucleotide 479. We think that the ATG in position 81 defines the initiation codon of the protein because it is surrounded by sequences which are in good agreement with the consensus sequence CA(G/A)CCATG for the initiation of translation in eukaryotes [20]. Moreover, the DNA sequences of the cDNA of clones cL71 and cL72 are highly divergent in the region upstream of the hypothetical translation initiation site, while they are practically identical in the rest of the sequence. These

21 3

21

111

201

291

381

471

561

65 1

-- c D N A cL71 1 GGCCTCATCCGTCATCCGTC c D N A cL72 --

MetAlaThrProArgSerALaLysLysALa ATCCGTCATCTTTGTGCTACAGCTTTACTCTCACTCCCCCTCCAGCCTACCCATGCAGCCATGGCTACTCCTCGCAGCGCCAAGAAGGCC

I ACCTCCTCTG C CCACACCC C T GCT TC TCT - - - A

ValArgLysSerGlySerLysSerAlaLysCysGlyLeuIlePheProValGlyArgValGlyGlyMetMetArgArgGlyGlnTyrAla GTCCGCAAGAGCGGCTCCAAGTCCGCGAAATGTGGTCTGATCTTCCCGGTGGGCCGCGTCGGCGGGATGATGCGCCGCGGCCAGTACGCT

C

A l a

ArgArgIleGlyAlaSerGlyAlaVatTyrLeuA~aA~aValLeuGluTyrLeuThrAlaGluLeuLeuAspLeuSerValLysAlaAla

CGCCGCATCGGTGCCTCTGGCGCCGTGTACCTGGCCGCCGTGCTGGAGTACCTGACGGCGGAGCTGCTGGAGCTGTCCGTGAAGGCGGCC

10

40

70

AlaGlnSerGlyLysLysArgCysArgLeuAsnProArgThrVaLMetLeuAlaAlaArgHisAspAspAspIleGlyThrLeuLeuLys 100 GCGCAGAGCGGGAAGAAGCGGTGCCGCCTGAACCCGCGCACCGTGATGCTGGCCGCGCGCCACGACGACGACATCGGCACGCTTCTGAAG

AsnValThrLeuSerHisSerGlyValValProAsnIleSerLysAlaMetAlaLysLysLysGlyG~yLysLysGlyLysA~aThrPro 130 AACGTGACCTTGTCTCACAGCGGCGTTGTGCCGAACATCAGCAAGGCGATGGCAAAGAAGAAGGGCGGCAAGAAGGGCAAGGCGACACCG

G G G G

S e r V a l V a 1 A r g S e r A l a * * * AGCGCGTAAGTCCTCCGGCCTGACAGCGCACACGCGCCGCTGTATTGTGCGCGTGCGCGCGGGTCCCGACTGGGGCCGGCGATGAGGCGC

A T C A T A C C T C C A T A G A G A C C C T A T C T T T T G T T T T A T G G C T T C T C A G A T G A C C A C T T G G T T C T T C C T G C C T T T G T T T G G T T T G T T T C T C T C

CTCCCCTCCGCCGAGGGTACGAGTCAGGGTAGGCTCGGACAAAAAAAAA

Fig. 1. Nucleotide sequence of the cL71 and cL72 cDNA clones. The deduced amino acid scquence is shown above each triplet of bases. The ochrc stop codon is marked by asterisks. The numbers at the left and at the right hand indicate the nuclcotide and amino acid positions, rcspectively. The vertical line at nucleotide position 26 indicates the beginning of the sequence of cL72 cDNA. The differences in nucleotide and amino acid sequence of cL72 cDNA have been located under cL71 cDNA. Hyphens under nucleotides at positions 73, 74, 75 and 516 indicate delctions of the scyuence of cL72 cDNA relative to cL71 cDNA. The arrow at position 174 indicates the 5’ end of cL7 cDNA. Nucleotide differences between cL71 and cL7 were not found.

Ldi MATPRB-AKKAVRKBG-SKSAKCGLIFPVGRVGGMMRRGQYARRIGABGA 4 8 H U SGRGKQGG..RA.AK-TR.BRA..Q......HRLL.K.N..E.V..GAP 4 8 spo .sGG~.~.A..~..AQ.~...A..A......~~L.K.N..Q.~..oAP 50

VYLAAVLEYLTAELLELSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTL 9 8 ............. I...AGN..--RDN.KT.II..HLQ..I.N.EELNK. 9 6 .......... A..I...AGN..--RDN.KT.II..HLQ..I.N.EELNK. 9 8

LXNVTLSHSGWPNISKAMAKKKGGKKGKATPSA .GK..IAQG..L...QAVLLP..TESHH..KGK .GH..IAQG......NAHLLP.QBG-...PSQEL

132 12 9 131

Fig.2. Maximal alignment of histone H2A sequences from L. d. in- ,fanturn (Ldi), human (Hum) 1371 and Schistosaccharomyccs pornbe (Spo) 1381. The entire L. d. infunturn sequence is presented. When amino acids are different in other organism, those are indicated. Dots represent amino acids which are the same as the Leishmania sequcnce. Gaps have been included (dashes) to maximal alignmenl.

features suggest, therefore, that the ORF2 codes for L. d. infaritum protein. Also we observed that ORF2 has a higher coding probability than the ORFl [19]. In fact, the ORF2 codes for a protein of 132 amino acids (Fig. 2) that shows 72.1 YO sequence similarity and 51.2% sequence identity with the histone H2A of Schizosaccharomycespombe (Fig. 2). Simi- lar percentages of sequence conservation were found when the deduced sequence of amino acids was compared with the human H2A histone sequence. Also, as it has been commonly observed in comparative analysis of the H2A sequences from various organisms [6], the polypeptide coded by the ORF2 shows a central core similar to that of the H2A proteins although with amino and carboxyl ends less conserved

(Fig. 2). Moreover, the protein coded by the ORF2 has a Lys-Lys-Lys motif in an equivalent position to the Lysl19 of the human histone H2A, which has been reported as the site of ubiquitin attachment [21]. All together, these data strongly indicate, therefore, that the polypeptide coded in the insert of the isolated cDNA clones is the H2A protein of L. (1. irzfantum.

Fig. 1 also shows that the deduced amino acid sequences from the cDNAs cL71 and cL72 are not the same due to the existence of five nucleotide transitions representing changes in codified amino acids. The 5’ untranslated regions (UTR) of these cDNAs are highly divergent in nucleotide sequence. These differences in sequence between the two cDNAs indicate that, at least, two H2A transcriptional units are synthesized during the promastigote stage of the parasite life cycle. In contrast, the 3’ UTR of both cDNAs is identical with the exception of a single nucleotide deletion at position 516 in the cL72 sequence. The sequence conservation of the 3‘ UTRs probably correlates with the potential functional role of that region. The 3‘ UTRs have a nucleotide sequence of dyad symmetry (nucleotides 673 - 690 of the cL71 cDNA sequence) which may form a stem-loop [22], similar in secondary structure to the stem-loops of histone mRNAs of higher eukaryotes. This stem-loop has been implicated in the regu- lation of protein expression [23].

Genomic organization of Leishmania histone H2A genes The Southern blots of genomic DNA probed with the cL7

cDNA revealed multiple hybridization bands for all restriction

214

Fig. 3. Genomic arrangement of the histone H2A genes in L. d. infunturn. Approximately 2 pg total DNA from promastigotes were digested with thc rcstriction enzymes HindIII (lane H), BamHI (lane B) and EcoRI (lane E) and separated on a 0.8% agarose gel. Following blotting, the filter was hybridized with the radiolabeled insert from cL7 clone. The numbers at the right indicate the sizes (in kb) and mobilities of the rcstriction fragments from HindIII-digested I DNA.

enzyme digestions used (Fig. 3) suggesting the presence of multiple H2A genes. Hybridization bands were obtained at 15.9,8.7,4.2 and 1.0 kb for HindIII; 27.9, 25.0 and 5.5 kb for BanzHI; and 19.9 and 12.7 kb for EcoRI. Since HindIII does not have cut sites in the nucleotide sequence of the cL71 cDNA, we think that there should be at least four H2A genes in the L. d. infantum genome and that, therefore, some of the genes separated by HindIII must be physically linked in the restriction fragments generated by BamHI and EcoRI. We cannot exclude, in addition, that in some of the large-size hybridization bands there could exist more than one gene organized in tandem as has been shown for the H2B genes of L. rnriettii [lo].

Expression of the L. d. infanturn H2A gene

When total RNA from promastigotes of L. d. infantum was probed with the insert of clone cL7 a single hybridization band of 0.7 kb was detected on Northern blots (Fig. 4). In contrast with most histone mRNAs from eukaryotes, except the variant histoncs of higher organisms [24, 251, all histone mRNAs from yeast cells [26], and the H3 and H4 histone mRNAs from many plants [27], the Leishmania H2A mRNAs are polyadenylated as deduced from the Northern blot analy- sis shown in Fig. 4A. In fact the three H2A cDNAs sequenced

Fig.4. Northern blot analysis of histone H2A mRNAs. (A, B) 10 pg non-polyadenylated RNA (lane l), 10 pg total RNA (lane 2), 2 pg polyadenylated RNA (lane 3) from L. d. infuntum promastigotes and 2 pg total RNA from T. cruzi epimastigotes (lane 4) were separated on 1 % agarose/formaldehyde gels and transferred to nylon filters. Filter A was hybridized with the radiolabeled insert of clone cL7 and filter B was hybridized using as probe the T. cruzi hsp70-coding DNA included in plasmid pBg2 [15]. (C, D) 2 pg total RNA from pro- mastigotes at either logarithmic growth phase (lane 1) or stationary growth phase (lane s) of L. d. infunturn were also fractionated in agarosc/formaldehyde gels and transferred to nylon filters. Filter C was hybridized with the same probe used for analysis of filter A. Filter D was hybridized with the probe used for analysis of filter B. Under C is shown the ethidium bromide staining of the RNA samples loaded. The positions and size of hybridizing bands (in kb) are indicated at the right.

show a poly(A) tail. Recently it has been reported that the histone H2B mRNA of L. enriettii is also polyadenylated [lo]. A similar-sized H2A mRNA species was found in a related parasite such as T. cruzi (Fig. 4A, lane 4). In order to deter- mine if the steady-state level of the H2A mRNA correlates with the rate of growth of the parasites, the RNA isolated from logarithmic and stationary growing parasites was probed with the cL7 clone. We observed that the level of H2A mRNA is significantly lower in parasites growing in stationary phase cultures (Fig. 4C, lane s ) than in the logarithmic phase (lane 1) in contrast with the steady-state level of the hsp70 mRNA (Fig. 4D) which does not show large variations in either stage. These results are an indication that the expression of the H2A genes is down-regulated in parasites growing in stationary phase, in agreement with the characteristic type of histone biosynthesis which is tightly linked to cellular DNA repli- cation [23].

21 5

Fig. 5. Identification of L. d. infanturn histone H2A labeled by a serum from a dog with visceral leishmaniasis 20 pg total proteins from induced cells carrying the recombinant plasmid pMS-cL7 (lane l), from induced cells carrying the pMS vector (lane 2) and from L. d. infanrzun promastigotes (lane 3) were resolved in a 12% polyacrylamide gel and electrophorctically transferred onto a nitrocellulose filter. (A) Ponceau S staining of filter. The Western blots were probed with either a canine leishmaniasis serum (B) and with the column-affinity-purified anti- serum against the pMS-cL7 fusion protein (C). Both sera were used at 1 :40 dilution. As second antibody, a 1 : 2000 dilution of peroxidase- immunoconjugate was used. The positions of the fusion protein from plasmid pMS-cL7 (F) and L. d. infunturn histone H2A (h2a) are indicated. At the left, the position and size (in kDa) of the SDS molecular mass markers (Sigma) loaded in the same gel are indicated.

Expression of the H2A cDNA and affinity purification of specific antibodies

Specific antibodies were purified from sera of infected dogs using the /l-galactosidase-fusion protein (expression product of pMS-cL7 plasmid) in order to identify in Western blot the L. d. infanturn protein band corresponding to the histone H2A. As expected, the SL9 serum recognizes only the expression product (Fig. 5 B) failing to detect the \i-galactosidase. Fig. 5 also shows that the purified antibodies recognize a single protein band of about 14 kDa which is the size of the protein codified by the nucleotide sequence of the cL71 clone. In some of the blots a protein band of 70 kDa was also labeled by the purified antibodies (Fig. 5B, lane 3). We think that this labeling is duc to the presence of a residual fraction of anti- bodies against the hsp70, given the strong reactivity of the original serum against this protein (laboratory data).

DISCUSSION

It has been reported that, during infection with several species of trypanosomes, a large proportion of the antibodies present in the sera of patients are essentially raised against the conserved members of the heat-shock protein family [2, 31. This feature is also characteristic of autoimmune diseases in which patients show high levels of autoantibodies against conserved proteins [28]. Since autoantibodies against the histone H2A protein have been, moreover, found in the sera of patients with systemic lupus erythematosus [29] and anti- histone antibodies have been described in mice affected with lupus [30], the finding of antibodies against the histone H2A in the sera of animals infected with Leishmania reinforces the hypothesis that a certain similarity might exist between the autoimmune process and parasite-induced diseases [31, 321. Whether or not this stimulation is correhted with specific characteristics of the proteins able to induce the autoimmune response is unknown but it should be noticed that, using statistical methods, Hrendel et al. [33] have detected that all of the nuclear and cytoplasmic autoantigens have segments highly rich in charged amino acids. In fact, the H2A histone protein is highly rich in basic amino acids. However, more efforts are necessary to understand the relationship between

parasite-induced and autoimmune diseases and the type of immunogenic stimulation which is responsible for the appear- ance of antibodies against conserved proteins in relation with the pathogenesis developed by the pathogens.

The data presented in this paper about the characterization of the histone H2A gene of Leishmania together with the recently reported data on the histone H2B of L. enriettii [lo] represent the first reports which demonstrate the existence of genes coding for histone proteins in kinetoplastid protozoa. It is likely that the histone H2A protein of Leishmania plays a role similar to that of eukaryotic organisms since the amino acid sequence of the histone H2A from this parasite shows a high structural conservation with the homologous histones of other eukaryotes [6]. In fact, the coded H2A protein of Leishmania is 132 amino acids long, which is the typical length for other H2A histones, and it has the central core and lysine motif typical of other histone H2As. However, despite the amino-acid sequence similarity existing between the H2A pro- tein deduced from clones cL71 and cL72 and the histone H2A from other organisms, the L. d. injantum H2A histone is relatively divergent in sequence. While the similarity between pairs of eukaryotic H2A proteins is close to 90% [6], the L. d. infanturn H2A histone shows a similarity level of about 70% with only a 50% of sequence identity with the H2A of eukaryotes. Most probably, this divergence is correlated with the phylogenetic distance between these parasites and the rest of the eukaryotes, including yeast [34]. Although the genes coding for histone proteins have not been characterized for parasites other than L. d. infanturn and L. enriettii, the exis- tence of histone or histone-like proteins has also been deter- mined electrophoretically in C. jasciculuta [9] and T. cruzi [8].

Our data also indicate that at least four genes coding for the H2A protein must exist in the genome of L. d. infhntum and that at least two of them are not identical in nucleotide sequence since cDNA clones have been isolated which differ in the DNA sequence corresponding to the 5' end and have nucleotide substitutions within the coding region reprcsenting changes in codified amino acids. Both gencs are, however, expresscd during the promastigote stage. The differences ob- served in the isolated H2A cDNAs of Leishmania may be correlated with the well known fact that the family of the H2A histones is unique among the other four core histone families

21 6

of the nucleosomes in containing three closely related subfam- ilies, conserved as separate entities during evolution. Since, however, the amino acid differences among the members of the H2A histone family are extensive [24, 35, 361 and the deduced amino acid differences between the two cDNAs that we have isolated are small, it is probable that both H2A- coding genes are not different members of the H2A histone family but that they arise from a recent gene duplication. At present we do not know whether or not the expression of each of the genes corresponding to the sequences of cL71 and cL72 are regulated in an identical manner since both genes differ significantly in their untranslated 5' end.

We thank Miguel Angel Fuertes for technical assistance. This work was supported by grant PB88-0011 and in part by LETI S. A. M. S. has a fellowship from LETI.

REFERENCES 1 . tIoward, J . G. (1985) in Leishmuniasis (Chang, K.-P. & Bray, R.

S., eds) vol. 1 , pp. 139 - 162. Elscvicr Science Publishing Co., New York.

2. MacFarlane, J., Blaxter, M. L., Bishop, R. P., Miles, M. A. & Kelly, J. M. (1990) Eur. J . Biochem. 190, 377-384.

3. Newport, G., Culpepper, J . &Agabian, N. (1988) Parasitol. Todiij

4. Pateraki, E.. Portocala, R., Labrousse, H. & Guesdon, J.-L.

5. Tan, E. M., Chan, E. K. L., Sullivan, K. F. & Rubin, R. L. (1984)

6. Wells, D. E. (1986) h'ircleic Acids Res. 14, rl19-rl49. 7. Astolfi Filho. S.. Martins de Sa, C. & Gander, E. S. (1990) Mol.

8. Toro. C. C. & Galanti. N. (1988) ESP. Cell Res. 174, 16-24. 9. Duschak. V. G. & Cazzulo. J. J. (1990) Biochirn. Biophw. Actu

1040, I59 - 166. 10. Genske, J . E., Cairns. B. R., Stack, S. P. & Landfear, S. M. (1991)

Mol. Cell. Bid. 11. 240 - 249. 11. Hyunh, T. V., Young. R. A. & Davis, R. W. (1985) in D N A

cloning (Glover, D., ed.) vol. 1, pp. 49 - 78, IRL Press, Oxford. 12. Ruther, U . & Muller-Hill, B. (1983) EMBO J . 2, 1791 -1794. 13. Carroll. S. B. & Laughon, A. (1987) in D N A cloning: u practical

upproucl7 (Glover. D. M., ed.) vol. 3, pp. 89-111, IRL Press, Oxford.

4,306-312.

(1983) Itlfi.cr. Immun. 42, 2-13.

Clin. Iintnunol. Immunoppath. 47, 121 - 132.

Rioclicvn. Purusitol. I , 45 - 53.

14. Laemmli. U. K. (1970) Nuture227, 680-685.

15. Requcna, J . M., Lopez, M. C., Jimenez-Ruiz, A,, dc la Torre, J .

16. Chomczynski, P. & Sacchi, N. (1 987) Anal. Biochom. 162, 1 56 -

17. Lchrach, H., Diamond, D., Wozney, J . M. & Boedtker, H. (1977)

18. Sangcr, F., Nicklen, S . & Coulson, A. R. (1977) Pruc. Null Acad.

19. Devereux, J. , Haeberli, P. & Smithies, 0. (1984) Nucleic Acids

20. Cavener, D. R. & Ray, S. C. (1991) Nucleic Acid.y Res. 19.3185-

21. Goldknopf, I. L. & Busch, H. (1977) Proc. Nut1 Acad. 5 i . USA

22. Soto, M., Requena, J. M., Jimcncz-Ruiz, A. & Alonso, C. (1991)

23. Schumperli, D. (1988) Trends Genet. 4, 187-191. 24. Mannironi, C., Bonner, W. M. & Hatch, C. L. (1989) Nucleic

25. Wells, D. & Kedes, L. (1985) Proc. Nut1 Acad. Sci. U S A 82,

26. Fahner, K., Yarger, J . & Hereford, L. (1980) Nucleic Acids Re.!.

27. Chaboute, M.-E., Chaubet, N., Clement, B., Gigot, C. & Philllips,

28. Guilbert, B., Dighiero, G. & Aurameas, S. (1982) J . Immunol.

29. Plaue, S., Muller, S. & van Regenmortel. M. H. V. (1989) J . E..wp. Med. 169,1607-1617.

30. Muller, S . , Couppez, M., Briand, J.-P., Gordon, J., Sautikre, P. & van Regenmortel, M. H. V. (1985) Biochim. Biophys. Actu827, 235 - 246.

C. & Alonso, C. (1988) Nucleic Acids Re.s. 16, 1393- 1406.

159.

Biochemistry 16,4743-4751.

Sci. USA 74, 5463 - 5467.

Res. 12. 387-395.

3192.

74, 864 - 868.

Nucleic Acids Res. 19, 4554 - 4554.

Acids Res. 17. 9113-9126.

2x34 - 2838.

8, 5725 - 5737.

G. (1988) Gene 71,217-223.

128,2179-2787.

31. Kierzenbaum, F. (1 986) J. Parasitol. 72, 201 - 21 1. 32. Ternynck, T., Bleux, C., Gregoire, J . , Avrameds, S. & Kanel-

33. Brendel, V., Dohlman, J., Blaisdell, B. E. & Karlin, S. (1991)

34. Sogin, M. L., Elwood, H. J . & Gunderson, H. H. (1986) Proc.

35. Challoner, P. B., Moss, S. B. & Groudine, M. (1989) Mol. Cell.

36. Cheng, G., Nandi, A,, Clerk, S . & Skoultchi. A. I . (1989) Proc..

37. Hayashi, T., Ohe, Y. , Hayashi, H. & Iwai, K . (1980) J . Biochem.

38. Matsumoto, S . & Yanagida, M. (1985) EMBO J . 4 , 3531 -

lopoulos-Langevin, C. (1990) J . Immunol. 144, 1504 - 151 1.

Proc. Nut1 Acad. Sci. USA 88, 1536 - 1540.

Nut1 Acad. Sci. USA 83, 1383 - 1387.

Bid. 9, 902-913.

Nut1 Acad. Sci. USA 86, 7002 - 7006.

( T o k ~ ~ o ) 88, 27 - 34.

3538.