Molecular and Biochemical Parasitology, 61 (1993) 265-274 265 © 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00

MOLBIO 02051

Isolation, characterization and analysis of the expression of the Leishmania ribosomal PO protein genes

Manue l Soto, Jose M. Requena and Carlos Alonso* Centro de Biologia Molecular "Severo Ochoa' ( CSIC-UAM), Universidad Autdnoma de Madrid, Cantoblanco, 28049 Madrid,

Spain (Received 16 February 1993; accepted 14 July 1993)

Two tandemly linked genes are present in the Leishmania infantum genome that code for the acidic ribosomal PO protein. The genes are identical in the coding region, although a striking lack of nucleotide sequence conservation is observed when the boundaries of the coding regions between both genes are compared. The 3' untranslated regions of the two genes are, moreover, different in size. The deduced amino acid sequence of the L. infantum PO protein (LiPO) shows a high degree of sequence conservation, including the highly charged conserved C-terminal domain, with the ribosomal PO proteins of other eukaryotic organisms. Northern blot experiments showed that two different size class transcripts are expressed in the gene cluster and that the steady state level of each of the transcripts in logarithmic phase promastigotes is markedly different. The abundance of both transcripts is down-regulated in parasite cultures on reaching stationary phase. Since it seems that the two Leishmania ribosomal PO genes are expressed in a single polycistronic transcript, it is likely that the different levels of PO mRNAs observed in cultured cells is due to a postranscriptional regulatory mechanism.

Key words: Leishmania infantum; Ribosomal PO protein; Tandemly linked genes; Differential gene expression

Introduction

Leishmania is a genus of parasitic protozoa capable of causing a spectrum of human and veterinary diseases [1]. Although during Leish- mania infection the mammalian immune system elicits high titers of circulating anti- bodies, it seems that these antibodies do not have an important effect on the resolution of an established infection [2]. However, the presence of specific immunoglobulins can be used for the isolation of many antigenic parasite proteins. Thus, as part of a strategy

~ p o n d i n g author.

Note: Nucleotide sequence data reported in this paper have been submitted to the EMBL data library with the accession number X72714.

Abbreviations: ORF, open reading frame; PCR, polymerase chain reaction; UTR, untranslated region.

leading to the identification of antigens of Leishmania infantum, we have screened a cDNA expression library using sera from infected dogs. As reported here, this strategy led to the isolation of cDNAs showing nucleotide sequence similarity with the genes coding for the ribosomal PO proteins [3].

The eukaryotic PO proteins are involved in the attachment to the ribosome of a set of highly acidic ribosomal proteins [4,5]. This complex of proteins, a general feature of the ribosomes from eukaryotic organisms, plays a role in the interaction between soluble factors and the ribosome during translational events [5,6]. The acidic ribosomal proteins and the ribosomal PO proteins present an almost identical highly charged conserved C-terminal domain [3,8]. The C-terminal domain has been described as being a strong antigenic determi- nant in certain autoimmune [9,10] and infec- tious diseases [11-14].

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Given the dual role that the ribosomal PO proteins must be playing in the Leishmania life cycle, as a regulatory factor of the translational machinery and as an antigen inducing the host humoral response, we have carried out mole- cular characterization of the genes coding for' this protein.

Materials and Methods

Parasites. Promastigotes of L. infantum (LEM 75; zymodeme 1) were grown at 26°C in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Flow Lab., Irvine, UK). Experi- mental cultures were initiated at 1 x 10 6

promastigotes m1-1 and subsequently har- vested for study at different points during their transition from logarithmic (5-9 x 106 promastigotes ml - l , days 2 - 3 ) p h a s e of .growth to the stationary (4-6 x 10"promas- tigotes ml -~, days 6-7) phase.

Screening of cDNA and genomic li- braries. The L. infantum cDNA expression library was made in 2gtl l as previously reported [15]. The immunological screening was carried out using a serum from a dog naturally infected with visceral leishmaniasis [15] using standard methodologies [16]. As secondary screening reagent, radioiodinated protein A (Amersham) was used. Four cDNAs, called L23, L25, L27, and L32, were isolated and subcloned into the EcoRI site of pUC 18 plasmid.

A L. infantum genomic DNA library was constructed using the 2EMBL-3 phage repla- cement vector. For that purpose the total parasite DNA was partially digested with Sau3A as described by Kaiser and Murray [17]. The 32p-labeled nick translated cDNA L27 was used as probe for in situ plaque hybridization [18]. From the positive phages isolated, one of them, called L27g-5, was chosen for a detailed restriction analysis.

Sequence determinations. DNA sequencing was conducted on double-stranded DNA by

the dideoxy chain termination method [19] using the Sequenase Kit (United States Biochemical Corp.). The nucleotide sequences of the cDNAs were determined in both strands using internal synthetic oligonucleotides.

The restriction map of the L27g-5 genomic clone was determined for a variety of restric- tion enzymes. The restriction fragments Sail- Sail of 0.6 kb (marked as I in Fig. IA), Sail- HindlII of 1.2 kb (II), HindlII-Sail of 1.5 kb ( l id and Sail-Sail of 1.8 kb (IV) were subcloned in the pUC18 cloning vector. Both strands of fragments II and III were sequenced using internal synthetic oligonucleotides, while fragments I and IV were partially sequenced. The analysis of the DNA and amino acid sequence was done using the University of Wisconsin Genetics Computer Group pro- grams [20] and by accessing to the GenBank and EMBL data bases of protein and DNA sequences.

Southern and Northern blot analysis. The L. infantum DNA and RNA were isolated as previously described [21,22]. Promastigote total DNA was digested with a variety of restriction enzymes, subjected to electrophor- esis in 0.8% agarose gels and transferred to nylon membranes (Hybond-N, Amersham) by standard procedures [18]. Total RNA was size separated on I% agarose-formaldehyde gels [23] and electrotransferred to nylon mem- branes using a LKB system (Pharmacia). Hybridizations, either for DNA or RNA analysis, were performed in 50% formamide/ 6 x SSC(1 x SSCis0 .15 MNaC1/0 .015M Na-citrate, pH 7.0)/0.1 SDS/0.25 mg ml - l herring sperm DNA at 42°C overnight. Final post-hybridization washes were performed in 0.1 x SSC/0.2% SDS at 50°C for 1 h. For reuse, blots were treated with 0.1% SDS for 30 min at 95°C to remove the previously hybridized probe. Removal of the probe was verified by autoradiography.

Amplification by the polymerase chain reac- tion. A purely coding DNA probe was synthesized by PCR amplification using as primers two oligonucleotides derived from the

coding regions of the LiPO genes. The sequence of the primers used was CPI: 5' TCGCGGGTGAACAGCACACC 3', and CP2: 5' GGTGGACAACTCGACGGCGA 3'. The 129-bp amplification product was called LiPO-CD (coding region). Also, a probe for the intergenic region was obtained by PCR amplification using the oligonucleo- tides CP3 (5' GTCTTCGGTGATGCTTT- CTC 3') and CP4 (5' GGGTTGTGGCACG- GACAATG 3'). This 274-bp amplification product was called LiPO-IN (intergenic re- gion), For PCR amplification the AmpliTaq DNA polymerase Kit (Perkin Elmer Cetus Corp.) was used, according to the manufac- turer's instructions. The final reaction volume was 50 /~1 with 0.3 /~g of the appropriate oligonucleotides added to the reaction mixture. The amplification cycle profile was: (a) denaturation at 94°C for 1 min; (b) annealing of primers for 1 min at 55°C; and (c) extension of primers for 1 min at 72°C.

Densitometric measurements. To quantify the intensity of the hybridization bands the autoradiographs were analyzed with a laser densitometer (Image Quant).

Results

Sequence analysis and genomic organization of L. infantum ribosomal PO protein genes. The screening of a 2gt 11 library with a serum from a dog naturally infected with L. infantum resulted in the isolation of several recombi- nant cDNA clones. The EcoRI inserts of the phage clones were subcloned in the pUC18 plasmid and sequenced. After a database search, four of the cDNAs revealed simila- rities with the carboxy-terminal coding region of the ribosomal PO protein gene family. Three of the cDNAs, called L23, L27 and L32, though different in size, showed the same nucleotide sequence. The fourth cDNA, called L25, showed differences in both the length and the nucleotid e sequence of the 3' untranslated region (UTR) compared with the other cDNAs. However, the sequence of the coding

267

region of L25 is identical to that of the other clones. The existence of two different popula- tions of cDNAs indicated that there must be at least two PO genes in the Leishmania genome. As none of the cDNAs contained the full coding region for the LiPO, the longest of the four characterized cDNAs, L27 cDNA, was used as probe to screen a L. infantum genomic 2-EMBL library. The genomic organization of the DNA region containing the LiPO gene deduced from the restriction and Southern analysis of the recombinant phage L27g-5, is shown in Fig. 1A. The relative autoradio-

A

sx B sH • ~ s ~ s s s

II I II I I 'Id ~ I I

Iv zz~ ~x z

B MSH M S H

Xb

- - 2 3 . 6

- - 8 . 6

- - 6 . 6

- - 4 . 3

-- 2 . 3

-- 2 . 0

Xb

- - 2 3 . 6

- - 8 . 6

-- G . 6

-- 4 . 3

-- 2 . 3

-- 2 . 0

L27 L25

Fig. 1. (A) Restriction map of the insert of the recombinant phage L27-g5. Restriction enzyme sites represented: S, Sail; M, Sinai; B, BamHI; E, EcoRI; H, HindllI. LiPO genes (A and B) are depicted as closed boxes: the black boxes indicate the coding regions and the cross-hatching boxes the 3' UTRs. Arrows above genes indicate the direction of transcription. Below the map the subcioned fragments used for sequence determinations are indicated. (B) L. infantum DNA was digested with Sinai (lane M), Sail (lane S) and HindlII (lane H) and analyzed by Southern hybridization with either L27 cDNA (left panel) or L25 eDNA (right panel) as probe. The sizes of DNA markers are indicated

on the right.

at•cgc•cgc•cgcgc•agagagcat•tatccctgcgtgccttcaatggagacttg•cacccctcttctct•ctctctgctt tctgctcc

• tcccc taa t tacc t tgac tgcc t t t tac t t • t t ccc t t tc ta t t tcc tc • • • t t t tggcaacc t tcc t ta tgcgcccaacacccacaac atacccacccacaaatcgt tgct tcacggcctcccctcgtgct t tgcagctccct t tagcaacg ATG CCG TCT ATC ACC ACT

GCC ARG CGC ATG GAC ARC ATG GGC ARG GCG ARG CAC ARC ARC GCT GCG ATT TCC TTC CAG GCG CTG AGC GTC TAC TAC CAG ACG GAG GAC GGC ATC CCG GTG GCG ACC CTG GCC GGC

GAG TAC GAG GAG CGC CTC GTC GTC CGC TCG CAG CAG GTG CAC ARG ACG CTG CAG GGC ARG ATC TTC ARC GAT CAG TGT GAG GAG GTC CAG GAG ATC ACG TCT GTG CCG TGT GAC GTG ATT GTC GCT CTG ARC ATT GCG ACG AAG ATT GGC GAC ARG GTG GAC ARC TCG GTG AAT GTG CTG TCC GTG TGG ATG GTG GAG ARG ATG CTG ATG ACG TCT TCG ACG ATT GGC CCG TCG TAC GAG TTC GAG GAG CAC TCT TGC TCG GCT GCT GCG GAG

90 180 262

CCC GCC GCT GCC GCG CCG GCC GCC CCT AGC GCC GCT GCC

AAG GAG GAG CCG GAG GAG AGC GAC GAG GAC GAC TTC GGC ATG GGC GGT CTC TTC TAR gcgactcgccatct 1230 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

ctta•cctcctt•tg•t•c•ctt•a••t•ctctc•ctctgcttctcctt•ca•t•tt••ct••ctct••c••gtat•t•tcgtc•catta 1320 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

cacccacctctcccacccctttgctctac•Cgctc•catgc•caatcc•tgaatcatcgagggaagtctctctgggtg•cagt•ggtaag 1410 ======================================== L27 cDNA ======================================== cttgt~aggaaa~ag~tgtgtgtgt~agcgg~caggtacgtcggaccacttaaacaaacaaacacacacacacac~gaaagactcacgta 1500

ca~catccgtccggcgcaacagcaacgtccgcc~c~c~aagcagagc~cgtgc~ctcattgtaccgct~tgaacgga~a~ggggggact 1590 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

c t t cgc t t t t t t c t t t t t c t t t t t t t t g t t t c •g tag t t t a t t c t t ca t t t t cc • t c t caac tcaaaaaaca •cacaaaaac •c • •aaac 1680 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

gcagcatgagtg•cgccgttgcaatcgg••acggt••c••c•caacgc•tcgtggcaactgcgcatgggttgctatct•at••at•gtt• 1770 cactgctgctcgaacaca•gtggacctccccccccccc•caacgacgacgtccggtcgagtcgcgggcgtgtggccgtgagcacagggta 1860 gcct t tc t t tgcgtcgcacagcacctatcgtcgtcgtcg•cactcctcatcacatc tccctcgtgtcgcacgaaggtgtgctgtc tg t •a 1950 •gacgcttccgtgtgagtaggtgcgtgcaaacatgcgt•catcggcaccggatcgcggtcg•gtaggttccacgctcctggagggtcgca 2040 agtgtct t~ctgctccag~tgactgatgaccaaggccatatcctcacgca~caccttcact~ctgcc~cgctgctt tcctccagcacg~a 2130 gc•agcacaggggcacgggt•ggggcggcaa•Cgagta•cctctgag•ttgtgcgtaggcgacacgtcgtgtgccagt•ggcactgcgc• 2220 cc t t t tcagt~ t t~ t~ tg tg~aacacag~tc~gcgcac~ct~ tc t tcggtgatgc t t tc tca t ta tgagccgct tgccgagcgtgc~cg 2310 c~accccc9~cccctcctcacctcctc~c~cg~agttaacgcgtgcacgctgtgtcccctgtgtaaagacagcttcccccacccccttgt 2400 caactccctc tc~gtccgtc t t tc~c~c~t tcat tc tc tc t tc t tcgtgaac~aaacacgaccactcgcctcgcatat tccgcgtgccca 2490 atatcccactcactcccttacacatgcattgtccgtgccacaaccc•gcgcacacttcggcacac•aaaaacaccttccccgaccccacg 2580 acagatagccaaggctattgcaagtctcacaag ATG CCG TCT ATC ACC ACT GCC AAG CGC GAG TAC GAG GAG CGC 2655

t~ W lit W W

CTC GTC GAC TGC CTG ACC AAG TAC AGC TGC GTG CAC GAT AAG ATC GTG GAG GAG TAC TCT GTG CTT GTC GCT GCT ARG ATT GCC AAC TCG ACG GTG TGG GAC

GTC GGC CGT GCG CTG CGC GCG GAG RAG CGC GCG CAR GCC ARG AAC CTG CTG AGC GGC ARC ACC GAC GCG CAC CGC GTG ARG CGC GGC AGC ACC GGC ATG GAG CCG ARG GGT ATG GTG GAG ATC GTG GCG ACG CTG CTG CAA AAG CTG CGC GGT GTG CTG TTC ACC CGC

CTG ATG GAR GGC CTG AGC ARC GTT GCG GCG GGC CCG ATG CTG GTG GAC GCC TTC ARG AAC

GTG CTG TTC GTG GGC ATG GAC AAC GTC CGC TCG CAG CAG 2724 AAG GCC GAG TTC ATG ATG GGC ARG ARG ACG CTG CAG GGC 2793 GAC GCG AGC CCC GAG GCG ARG CAC TTC ARC GAT CAG TGT 2862 GGC CTC ATC TTC ACG ARC ARC GCT GTC CAG GAG ATC ACG 2931 GCG GCG CGT GTC GGA GCG ATT TCC CCG TGT GAC GTG ATT 3000 ACC CAG ACG TCC TTC TTC CAG GCG CTG ARC ATT GCG ACG 3069 ACG GAG ARG ARG GTG CTG AGC GTC GGC GAC AAG GTG GAC 3138 AAC ATC AGC CCG TTC TAC TAC CA t GTG ART GTG CTG TCC 3207 GAG GAC CTG ATG ATG ACG GAG GAC ATG GTG GAG AAG ATG 3276 ATG GCG CTG GGT GCT GGC ATC CCG ACG TCT TCG ACG ATT 3345 CTG CTG GCT GTC TCT GTG GCG ACC TCG TAC GAG TTC GAG 3414

3483 GAG CAC ARC GGC AAG GAG CTG CGC GAG GCC GCG ATC ARC GGC CTG CTG GCC GGC TCT TGC TCG GCT GCT

GCG GAG CCC GCC GCT GCC GCG CCG GCC GCC CCT AGC GCC GCT GCC ARG GAG GAG CCG GAG GAG AGC GAC 3552 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = L25 cDNA ========================================= GAG GAC GAC TTC GGC ATG GGC GGT CTC TTC TAA 9cgactcgccatctcccactgagcaccgtcgagtgt tcgtgtgt tc 3631

•c••••t••aca•c•gc•a•c•tgt••t•ccctt••atcatca••aa•ca•ctctctccctttctctct•t•ttcttcgtttcttc•ttc 3721

at tagt t t tggatcgccgtgcgctgcgcatcgctcagt tc tcat t ta ta tcaataacaacaacgaagac 3790

GAC TGC CTG ACC AAG TAC AGC TGC GTG CTG TTC GTG GGC 331 GAT GTC GGC CGT GCG CTG CGC GCG AAG GCC GAG TTC ATG 400 GTG GAG AAG CGC GCG CAR GCC RAG GAC GCG AGC CCC GAG 469 TAC ARC CTG CTG AGC GGC ARC ACC GGC CTC ATC TTC ACG 538 CTT GAC GCG CAC CGC GTG ARG CGC GCG GCG CGT GTC GGA 607 GCT GGC AGC ACC GGC ATG GAG CCG ACC CAG ACG TCC TTC 676 GCC ARG GGT ATG GTG GAG ATC GTG ACG GAG ARG ARG GTG 745 AC t GCG ACG CTG CTG CA&, RAG CTG AAC ATC AGC CCG TTC 814 GAC CGC GGT GTG CTG TTC ACC CGC GAG GAC CTG ATG ATG 883 GAA GGC CTG AGC ARC GTT GCG GCG ATG GCG CTG GGT GCT 952 ATG CTG GTG GAC GCC TTC RAG ARC CTG CTG GCT GTC TCT 1021 ARC GGC ARG GAG CTG CGC GAG GCC GCG ATC AAC GGC CTG 1090

1159

graphic intensity observed after Southern blot analysis of total DNA digested with SalI (Fig. 1B) and hybridization with either the L25 or L27 cDNA probes confirmed that at least two PO genes must exist in the Leishmania genome. It may be observed that the L27 cDNA highly labels the 2.4-kb SalI band while the L25 cDNA labels with higher intensity the 1.5-kb- SalI band. The specific labeling of the SalI bands by each of the probes must be attributed to the differences in the 3' UTRs between the cDNAs. Since the genomic hybridization pattern using as probe the L27 cDNA coincided with that predicted from the map of the L27g-5 phage (Fig. 1A), we may also conclude that there are no other LiPO genes in the Leishmania genome. As expected, we have observed that the L27 cDNA probe maps in a single chromosome in L. infantum (data not shown).

Sequence analysis. The nucleotide sequence determination of four restriction fragments (I- IV) from phage L27g-5 (Fig. 1A) showed that there are two genes coding for the Leishmania PO protein (Fig. 2), since the sequenced fragment contains two identical open reading frames (ORFs) of 969 nucleotides in length that show high similarity with the nucleotide sequence of the PO genes [3]. The two genes are head-to-tail tandemly linked and were desig- nated LiPO-A and B.

Nucleotide sequence comparison between the LiPO-A and the LiPO-B genes indicated that the two genes share the same nucleotide sequence in the coding region. However, a marked lack of conservation in the sequence of the two genes is observed upstream of the initiation codon. Also, a sudden loss of sequence identity occurs 15 nucleotides down- stream of the termination codon. Since the four cDNAs have a 3' poly(A) tail and it is

269

added at the same position in the three cDNAs derived from the LiPO-A gene (L23, L27 and L32), it was possible to define the length of the 3' UTRs. Thus, while the 3' UTR of gene LiPO-A is 448 nucleotides in length, the 3' UTR of gene LiPO-B has 163 nucleotides. The only remarkable feature detected in the 3' UTRs is the presence, in the LiPO-A gene, of a (GT)5 sequence localized in position 1426 and a (CA)6 sequence located 37 nucleotides downstream of the (GT)5 which may form a stem loop structure (Fig. 2A).

Deduced amino acid sequence of the LiPO protein. The deduced L. infantum ribosomal PO protein has a length of 323 amino acids (Fig. 3A), with a size of 34.87 kDa and an isoelectric point of 4.93. The comparison of the amino acid sequence of LiPO with other ribosomal PO proteins reveals a significant sequence conservation, reaching 79.5% se- quence similarity (64.59% sequence identity) when aligned with the T. cruzi ribosomal PO protein [13,24]. The structure of the Leishma- nia LiPO protein resembles that previously reported for the PO proteins from other eukaryotic organisms. The C-terminal domain preceded by an alanine- and proline-rich hinge region, characteristic of both eukaryotic ribosomal PO proteins and the 13-kDa species of the acidic ribosomal proteins [3,8], is also found in the Leishmania PO protein. The LiPO protein also displays the arginine- and lysine- rich region (residues 40-70) that has been shown to be involved in the interaction with the 28S rRNA [25]. The protein also has a region (residues 200-280) capable of adopting a putative hydrophobic zipper structure which may interact with the 13-kDa acidic ribosomal proteins [26]. However, as it has been also reported for the TcPO [24], the hydrophobic C- terminal F G M G G L F sequence of LiPO is

4----

Fig. 2. Nucleotide sequence of the LiPO gene cluster. The coding regions are indicated by capital letters separated in triplets. The nucleotide sequence spanned by the L27 and L25 cDNAs are double underlined and the locations of the 3' poly(A) tails are indicated by AAAAA >. The underlined nucleotide sequences correspond to the amplification products LiPO-CD (coding regions) and LiPO-IN (intergenic region). Putative splice acceptor sites AG are marked by asterisks. The (GT)5 and (CA)6

sequences are also underlined.

270

A

ToP0

LiP0

B

MPSVS~YRERFNGCLTKYGRVLFCLMDNVRSQQVHDVRRDLRGLGE III: IIIIIIII: :IIIII: III IIIIIIIIIIII I II: :I MPSZTTAEREYEERLVDCLTKYSCVLFVGMDNVRSQQVHDVG~

LVMGKKTLQKKXVERRAEDKKASAYDKLLYNTCIEFdKLLCGNTALIFTNE ::IIIIIII IIII:II: I II: I : : I I II:III:IIIII: FMMGKKTLQGKIVEERAQAKDASPEAEHFNDQCEEYNLLSGNTGLIFTNN

EXPVXTAVLDKHRVQAPARVGP-SPMRRHCPAGNTGMIPKATSFFQALNI : II III III :IIII: II :II IIIII IIIIIIIII

AVQEITSVLDAHRVKRAARVGAISPCDVlVAAGSTGMRPTQTSFFQALNI

ATKIAKGTVEIVSDKKVLSVGDRVDNSTATLLQKLDXSPFYYQVEVQSVW IIIIIII IIII :IIIIIIII:IIIIIIIIIIII:IIIIIIII:I III ATKIAKGMVEIVTEKKVLSVGDKVDNSTATLLQKLNISPFYYQVNVLSVW

DRGMLFLREDLSITDDVVEKYLLEGISNVAALSLGAGIPTAATLPHMIMD III:II I I I I :I:I:III I:II:IIIII: IIIIIII I: I::I DRGVLFTREDLMMTEDMVEEMLMEGLSNVAAMALGAGIPTSSTIGPMLVD

AFKTLLGASVATEYEFDEFDGKNLRKAALEGNLGGGVADAAAAADTGAAA III II: IIII III:I :II:II II::I I:I: II :I ::II AFKNLLAVSVATSYEFEEHNGKELREAAINGLLAGSCSAAAEPAAAAPAA

APAAAAEPEEEDDDDDFGMGALF 322 : I I I I I I I:111111:11 PSAAAKEEPEESDEDDFGMGGLF 323

50

50

1 0 0

100

149

150

199

2 0 0

2 4 9

250

299

300

HsPO ScPO TcPO LiPO HmPO

KEESEESDED MGFGLFD AEEEEESDDD MGFGLFD EEEDDDDDFG MG-ALF- PEESDEDDFG MG-GLF- DDDDEDAGDA LG-AMF-

Fig. 3. (A) Predicted amino acid sequence of the Leishmania PO protein (LiPO) and its comparison with the T. cruz iPO protein (TcPO) [13]. Vertical lines indicate identity of sequence and double dots indicate conservative changes. Gaps ( - ) have been introduced to maximize the homology. (B) Sequence comparison of the carboxy terminal residues of PO proteins from different organisms: HsPO, human PO [4]; ScPO, S. cerevisiae [33]; TcPO, T. cruzi PO [13]; LiPO, L. infantum PO (this work);

HmPO, Halobacterium marismortui L10 [34].

more similar to the equivalent sequence of the archaebacterial HmPO protein than to the eukaryotic consensus sequence M G F G L F D (Fig. 3B).

Expression of L. infantum ribosomal PO genes. Northern blot analysis using total RNA has revealed that two transcripts of about I. 1 and 1.4 kb are expressed in the LiPO

gene cluster (Fig. 4A). An identical hybridiza- tion pattern was obtained when poly(A) RNA was used (data not shown) indicating that both transcripts are polyadenylated. The difference in size of the transcripts is in agreement with the deduced size of the two LiPO genes, considering that the 3' UTR of gene B is approximately 300 nucleotides shorter than that of gene A (Fig. 2). The assignment of each of the genes to the transcripts was further demonstrated by Northern blot experiments using either the L27 cDNA or the L25 eDNA.

A

1 2 3 4

3.9 - -

2 .3 - -

- - 1.4 - -

5 -i B C

1 s 1 s

1 . 4 - -

1 . 1 - -

2 . 0

E J [ I . I ~ L

Fig. 4. (A) Total RNA was analyzed by northern hybridization using as probes the L27 cDNA (lane 1), L25 eDNA (lane 2), LiPO-CD (lanes 3 and 4) and LiPO-IN (lane 5). The exposure times for lanes 1, 2 and 3 were identical. The hybridization blot shown in lane 4 is the same as that of lane 3 but with a 5-fold exposure time. The molecular sizes of the transcripts are indicated in kb. (B) A Northern blot containing total RNA isolated from either logarithmic phase promastigotes (lane 1) or stationary phase promastigotes (lane s) was hybridized with the LiPO- CD probe. After autoradiographic exposure, the filter was rehybridized with a T. cruzi 18S rDNA probe to reveal any differences in loading RNA amounts between tracks. (C) After removing the probe, the Northern blot shown in B was hybridized using the T, cruzi ct-tubulin eDNA of clone

pTcct3 [35].

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Densitometric analysis showed that the L25 cDNA probe labels with significantly higher intensity the 1.1 kb than the 1.4 kb transcript while the L27 cDNA probe labels the 1.4 kb transcripts with 1.5-fold higher intensity than the 1.1 kb transcript (Fig. 4A). These results are an additional evidence that the 1.4-kb and 1.1-kb transcripts are expressed from the LiPO-A and LiPO-B genes, respectively. In order to determine the relative abundance of the two transcripts, the Northern blots were hybridized with the LiPO-CD amplification product (this DNA fragment spans only part of the coding region). The results showed that the 1.1 kb long transcript is 6-fold more abundant than the 1.4-kb transcript (Fig. 4A). The high abundance of the 1.1-kb transcript explains why the L27 cDNA probe labels with similar intensity both RNA bands although it is transcribed from gene A. In addition to the 1.1-kb and 1.4-kb transcripts, two additional transcripts of 2.3 and 3.9 kb were evidenced upon longer exposure of the filter. In order to analysis the nature of these higher-molecular weight transcripts, the same Northern blot was hybridized with a probe derived from the intergenic region (LiPO-IN; see Fig. 2 for location of the probe). Since this probe hybridizes with the 3.9-kb transcript (Fig. 4A) it must be considered as an intermediate of the polycistronic transcription of the LiPO gene cluster. Thus, considering the polycistronic transcription, the differential accumulation of the mRNAs is an indication of the different processing and stability rates of the 1.1-kb transcript relative to that of the 1.4- kb transcript.

We have also analyzed the distribution of the two transcripts in the different growth phases of cultured L. infantum promastigotes (Fig. 4B). Since the abundance of the LiPO RNA is about 6-fold higher in logarithmic phase promastigotes than in stationary phase (metacyclic) promastigotes, it is likely that a down-regulation of the LiPO gene expression occurs during the stationary phase of Leish- mania promastigotes (Fig. 4B). Furthermore, since the abundance of ~-tubulin transcripts are similar in both phases of growth (Fig. 4C),

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the down-regulation of the LiPO expression does not appear to be a direct consequence of a less active metabolic state of the stationary phase parasites. In fact, down-regulation mechanisms have been also described for the yeast ribosomal PO genes [8]. However, as expected from a polycistronic transcript, the decrease in RNA levels of both transcripts occurring during the stationary phase does not affect the 6:1 relative ratio between the two mRNA populations.

Discussion

The presence of a set of very acidic ribosomal proteins in the large ribosomal subunit during translational events is a common feature of the ribosomes of all organisms studied so far. The acidic riboso- mal proteins are anchored to the ribosomes as a complex of two dimers mounted on a single protein termed ribosomal PO protein. Although the eukaryotic acidic ribosomal proteins and the ribosomal PO proteins possess different amino acid sequences and molecular weights they share a conserved C- terminal domain rich in charged amino acids [3,4,8]. This conserved domain has been described as an antigenic determinant in some autoimmune diseases such as systemic lupus erythematosus [9,10], and also in infectious diseases produced by T. cruzi [11,13,14,24] and Leishmania [12].

In the present paper we have shown that the Leishmania ribosomal PO protein possesses the structural characteristics previously reported for the eukaryotic PO proteins. The universal conserved antigenic C-terminal domain pre- ceded by an alanine- and proline-rich hinge. region is also present in the Leishmania PO protein. Nevertheless, as it has been reported for the TcPO protein, the last 7 residues of the C-terminal of the LiPO protein slightly differs from the eukaryotic consensus sequence [13].

The analysis of the genomic organization of the Leishmania ribosomal PO genes showed the existence in the genome of two genes organized in a tandem array. The absolute

degree of conservation in the coding region of both genes contrasts, however, with the high diversity of the sequence of the 5' and the 3' UTRs of the genes. Sequence divergence within the UTRs of repeated genes, located in the same tandem array, have been described for other genes of trypanosomatids [27,28]. The sequence of the 3' UTR of the genes from these parasites seems to play an important role in the regulation of gene expression due to the presence of sequence elements that can form secondary structures in the mRNA [29-31].

In the LiPO gene cluster both genes, which are constitutively expressed during the pro- mastigote stage, are transcribed as two well- defined size-class mRNAs. Besides, the steady- state level of each one of the transcripts is markedly different. Given that both genes are transcribed as a polycistronic precursor, the differences in mRNAs levels must be explained in terms of stability of the mRNAs and/or maturation of the polycistronic precursors. Thus, it is probable that the regulatory elements responsible for the differences in processing and stability rates of the LiPO transcripts locate within, or close to, the divergent UTRs. A search for the function of these putative sequence elements is being done using appropriate transfection vectors [32].

Acknowledgements

This work was supported by grants 160/9 and SAF93-0146 from Plan Regional de Investigaci6n de la Comunidad Aut6noma de Madrid and CICYT. The support of LETI, S.A. and the institutional grant of the Funda- ci6n Ram6n Areces are also gratefully ac- knowledged. M.S. is supported by a doctoral fellowship from Comunidad Aut6noma de Madrid. We thank Dr. E. Rondinelli for the ~-tubulin cDNA clone.

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