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Molecular Characterization of the Leishmania braziliensis L6 Ribosomal ProteinAuthor(s): M. C. Thomas, E. Martinez-Carretero, E. Carmelo, A. C. González, and B. ValladaresSource: Journal of Parasitology, 90(4):908-913. 2004.Published By: American Society of ParasitologistsDOI: http://dx.doi.org/10.1645/GE-3297RNURL: http://www.bioone.org/doi/full/10.1645/GE-3297RN

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RESEARCH NOTES 907

J. Parasitol., 90(4), 2004, pp. 907–908q American Society of Parasitologists 2004

Fatal Toxoplasmosis in a Bald Eagle (Haliaeetus leucocephalus)

K. A. Szabo, M. G. Mense, T. P. Lipscomb*, K. J. Felix†, and J. P. Dubey‡§, Department of Veterinary Pathology, Armed Forces Institute ofPathology, Washington, D.C. 20306-6000; *6 Aura’s Way, Bow, New Hampshire 03304; †Glenwood Pet Hospital, 3853 Peach Street, Erie,Pennsylvania 16509; ‡Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center,United States Department of Agriculture, Beltsville, Maryland 20705-2350; §To whom correspondence should be addressed. e-mail:jdubey@anri.barc.usda.gov

FIGURE 1. Toxoplasma gondii and associated lesions in the heart of the naturally infected eagle (A–C, HE; D, B-H Gram stain). A. Aninflammatory focus with central necrosis (arrow). Bar 5 1,000 mm. B. Higher magnification showing severe myocarditis. Bar 5 300 mm. C.Tachyzoites (arrows) among degenerating myocardial myocytes. Bar 5 40 mm. D. A group of tachyzoites (arrow). Bar 5 0.9 mm.

ABSTRACT: Toxoplasma gondii tachyzoites were identified in the myo-cardium of a bald eagle (Haliaeetus leucocephalus) that died of nec-rotizing myocarditis. The diagnosis was confirmed by immunohisto-chemical staining with T. gondii–specific polyclonal antibodies. This isa new host record for T. gondii.

Toxoplasma gondii infections have been reported in many species ofwarm-blooded animals including birds (Dubey and Beattie, 1988; Dub-ey, 2002). Certain passerine birds are highly susceptible to clinical toxo-plasmosis. Recently, Dubey (2002) summarized subclinical and clinicalT. gondii in all avian species. The objective of the present article is todocument clinical T. gondii infection in a bald eagle (Haliaeetus leu-cocephalus) for the first time.

An immature male bald eagle was found in distress in water with noevidence of injury. On physical examination, the bird was normal butweak and responded initially to supportive care, including fluid therapyand force-feeding, for approximately 1 wk. The eagle developed respi-ratory distress, which resulted in death on 29 October 2002. Necropsywas performed 2 days later. The eagle weighed 3.5 kg, had a greenright-leg band inscribed B06B06, and a silver left-leg band inscribed62945823. The proventriculus contained fish ingesta and grit. The no-table gross lesions included an enlarged and mottled liver, congestedlungs, and an unidentified nematode in the small intestine.

Specimens were fixed in 10% formalin and submitted to the ArmedForces Institute of Pathology, Washington, D.C., for histologic exami-nation. The formalin-fixed tissues were embedded in paraffin, and 5-

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mm sections were placed on microscopic glass slides and stained withhematoxylin and eosin (HE), Giemsa, Ziehl–Neelson acid fast, Brownand Brenn and Brown–Hopps (B-H) Gram stains, Gomori methenaminesilver (GMS) and periodic acid–Schiff reaction (PAS) using routine pro-cedures. The tissues were then examined microscopically. Deparaffini-zed sections of heart were stained with T. gondii, Sarcocystis neurona,and Neospora caninum polyclonal rabbit antibodies as described (Lind-say and Dubey, 1989; Dubey and Hamir, 2000; Dubey et al., 2001).Sections of heart were also stained with BAG-1 antibodies against T.gondii bradyzoites (McAllister et al., 1996). The BAG-1 antibodies arebradyzoites specific and do not react with tachyzoites (McAllister et al.,1996).

Microscopically, the most marked lesion was the multifocal and ex-tensive necrotizing and lymphoplasmacytic myocarditis (Fig. 1A–C).There was perivascular cuffing of coronary vessels by lymphocytes,plasma cells, macrophages, and eosinophils. In the areas of necrosis,the cardiomyocyte sarcoplasm contained groups of protozoa (Fig. 1C).The protozoan groups were up to 20 mm in diameter and contained upto 30 zoites (Fig. 1D). These protozoa stained faintly with HE, andprominently with Giemsa, GMS, B-H Gram stain, Ziehl–Neelson acidfast, and PAS.

Other histopathologic findings included mild splenic lymphoid ne-crosis, mild lymphoplasmacytic and necrotizing periportal hepatitis, andmultifocal lymphoplasmacytic bronchopneumonia. The esophagus, pro-ventriculus, small intestine, kidney, and pancreas were within normallimits.

The protozoans stained positively with T. gondii but not with N.caninum or S. neurona polyclonal antibodies. A few groups of proto-zoans reacted positively with BAG-1 antibodies, indicating the presenceof bradyzoites.

The bald eagle is a protected species and belongs to the order Fal-coniformes, family Accipitriformes. Among all avian species, birds ofthe family Accipitriformes are more resistant to clinical toxoplasmosisthan other birds, although they can harbor viable T. gondii (Dubey,2002). Viable T. gondii was isolated from tissues of hawks (Accipitergentites, A. cooperi, Bueto jamaicensis, and B. lineatus), common buz-zard (B. buteo), kestrel (Falco tinnunculus, F. sparverius), and harrier(Circus macrourus) (Pak, 1976; Literak et al., 1992; Lindsay et al.,1993). Protozoan encephalitis associated with a Sarcocystis sp. was re-ported by Aguilar et al. (1991). In the present study, the diagnosis oftoxoplasmosis was made on the basis of structure and antigenicity of

the parasite. The parasite divided by endodyogeny, and it stained pos-itively with T. gondii–specific antibodies. The parasite did not stain withS. neurona or N. caninum antibodies. This is a new host record for T.gondii and should be considered in the differential diagnosis of necro-tizing myocarditis in eagles.

The authors thank Sean Hahn for technical assistance.

LITERATURE CITED

AGUILAR, R. F., D. P. SHAW, J. P. DUBEY, AND P. REDIG. 1991. Sarco-cystis-associated encephalitis in an immature northern goshawk(Accipiter gentilis atricapillus). Journal of Zoo and Wildlife Med-icine 22: 466–469.

DUBEY, J. P. 2002. A review of toxoplasmosis in wild birds. VeterinaryParasitology 106: 121–153.

———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man.CRC Press, Boca Raton, Florida, 220 p.

———, M. M. GARNER, M. M. WILLETTE, K. L. BATEY, AND C. H.GARDINER. 2001. Disseminated toxoplasmosis in magpie geese (An-seranas semipalmata) with large numbers of tissue cysts in livers.Journal of Parasitology 87: 219–223.

———, AND A. N. HAMIR. 2000. Immunohistochemical confirmation ofSarcocystis neurona infections in raccoons, mink, cat, skunk andpony. Journal of Parasitology 86: 1150–1152.

LINDSAY, D. S., AND J. P. DUBEY. 1989. Immunohistochemical diagnosisof Neospora caninum in tissue sections. American Journal of Vet-erinary Research 50: 1981–1983.

———, P. C. SMITH, F. J. HOERR, AND B. L. BLAGBURN. 1993. Preva-lence of encysted Toxoplasma gondii in raptors from Alabama.Journal of Parasitology 79: 870–873.

LITERAK, I., K. HEJLICEK, J. NEZVAL, AND C. FOLK. 1992. Incidence ofToxoplasma gondii in populations of wild birds in the Czech Re-public. Avian Pathology 21: 659–665.

MCALLISTER, M. M., S. F. PARMLEY, L. M. WEISS, V. J. WELCH, AND A.M. MCGUIRE. 1996. An immunohistochemical method for detectingbradyzoite antigen (BAG 5) in Toxoplasma gondii–infected tissuescross-reacts with a Neospora caninum bradyzoite antigen. Journalof Parasitology 82: 354–355.

PAK, S. M. 1976. Toxoplasmosis of birds in Kazakhstan. Nauka Pub-lishing Alma-Alta, USSR, 115 p.

J. Parasitol., 90(4), 2004, pp. 908–913q American Society of Parasitologists 2004

Molecular Characterization of the Leishmania braziliensis L6 Ribosomal Protein

M. C. Thomas, E. Martinez-Carretero*, E. Carmelo*, A. C. Gonzalez*, and B. Valladares*† , Instituto de Parasitologia y Biomedicina LopezNeyra, C.S.J.C. Av. del Conocimiento s/n 18100 Granada, Spain; *Facultad de Farmacia, Departamento de Parasitologıa, Ecologıa y Genetica,C/ Astrofısico Francisco Sanchez s/n, Universidad de La Laguna, Tenerife, Spain; †To whom correspondence should be addressed. e-mail:bvallada@ull.es

ABSTRACT: By screening a Leishmania braziliensis complementaryDNA library with a pool of sera from leishmaniasis patients, the genecoding for L6 ribosomal protein was isolated. The sequence, genomicorganization, and transcription of this gene are described in this article.The sequence analysis of the L. braziliensis L6 gene shows a singleopen reading frame, which codes for a protein of 192 amino acids (aa)with a hypothetical molecular mass of 20.9 kDa. The protein exhibitssignificant sequence similarity to L6 ribosomal proteins from highereukaryotes and yeast. Thus, the L. braziliensis L6 protein contains 4functional motifs, which are located at equivalent positions in other L6ribosomal proteins described previously. Interestingly, the L6 ribosomalprotein from L. braziliensis contains a specific region of 14 aa and atyrosine kinase motif, which is absent in human and C. elegans L6protein. The locus coding the L. braziliensis L6 ribosomal protein isformed by 2 gene copies arranged in tandem and located in a chro-mosome of approximately 0.9 Mb. The genes are actively transcribed

as 2 polyadenylated transcripts of approximately 1.15 and 0.85 kb,which differ in their steady-state level and stability.

Several species of Leishmania are etiological agents for a wide spec-trum of severe human diseases that affect more than 12 million peoplethroughout the world. The parasite is transmitted by the bite of an in-fected female sand fly. There are about 30 species of sand flies, whichcan transmit at least 20 different Leishmania species. These parasitesconstitute an additional health danger for the immunosuppressed pop-ulation. Thus, for example, Leishmania spp.–human immunodeficiencyvirus coinfection may cause serious health problems through a syner-getic effect on the infection of the other (Wolday et al., 1999). Leish-mania braziliensis is one of the major causative agents of mucocuta-neous leishmaniasis or New World leishmaniasis, which occurs in Cen-tral and South America. The promastigote form of the parasite becomesinfective before entering the host macrophage. This process is mimicked

RESEARCH NOTES 909

FIGURE 1. Nucleotide and deduced aa sequences of the L. brazilien-sis L6 protein. Numbers to the left of the sequence indicate the nucle-otide position. The spliced leader sequence is underlined. The L6 startcodon is shown in bold and the stop codon marked by an asterisk.

by the promastigote in liquid culture medium, developing from a non-infectious form during logarithmic growth to a highly infectious format the nonreplicative stationary phase (reviewed in Handman, 2000).

Leishmania species and other related trypanosomatids share in com-mon several unusual biological features, i.e., polycistronic transcription,genes organized in tandem arrays (Munich and Boothroyd, 1988),transsplicing of precursor RNAs (Murphy et al., 1986; Sutton andBoothroyd, 1986), and posttranscriptional editing of mitochondrial tran-scripts (Simpson and Shaw, 1989), which could be targets for specificdrug therapy. However, the available chemotherapeutic drugs againstleishmaniasis have a limited efficacy because they are relatively toxic,and resistant parasites are frequently observed (Borst and Quellete,1995; Boelaert et al., 2000).

Ribosomes are the protein-synthesizing machinery in all living cells.In Leishmania spp., ribosomes are the targets for an aminoglycosideantibiotic, paromomycin (Maarouf et al., 1995). The ribosomal L6 pro-tein from higher eukaryotes is located in the large ribosomal subunitthat occupies a central position in the ribosomal structure. It is closelyadjacent to 28S ribosomal RNA (rRNA) but also adjacent to the 18SrRNA across the ribosomal interface (Nygard and Nika, 1982). It spe-cifically binds to the Tax responsive element of human T-cell leukemiavirus type I (Morita et al., 1993). The expression of L6 gene is consti-tutive and ubiquitous, which argues for an important and biologicallyconserved function (Nacken et al., 1995).

In this article, we describe the isolation of a complementary DNA(cDNA) coding for the L. braziliensis L6 ribosomal protein, as well assome features regarding genomic organization, gene expression, andstability of the transcripts. The deduced amino acid (aa) sequence fromL. braziliensis L6 ribosomal protein possesses some domains that arehighly conserved among eukaryotic organisms. Interestingly, the L. bra-ziliensis protein has an additional region of 14 aa located in the centralpart of the protein and a tyrosine kinase motif, which is absent in humanand C. elegans L6 protein.

Promastigotes of L. braziliensis (MOHN/PE/95/LQ-8) were grownwith gentle shaking at 22 C in Rosewell Park Memorial Institute 1640medium (GIBCO, Paisley, U.K.) supplemented with 20% (v/v) heat-inactivated fetal bovine serum. Experimental cultures were initiated at1 3 106 promastigotes ml21 and subsequently harvested at 2 differenttimes during their transition from logarithmic to the stationary growthphase.

Total RNA from promastigotes of L. braziliensis in the logarithmicgrowth phase was purified using QuickPrep micro messenger RNA(mRNA) purification kit (Amersham Biosciences, Buckinghamshire,U.K.). cDNA was synthesized using the TimeSaver cDNA SynthesisKit in combination with the Directional Cloning Toolbox (PharmaciaBiotech, Cambridge, U.K.) and cloned into pBluescript KS(2) phagem-id vector (Stratagene, La Jolla, California). This library was subsequent-

ly introduced into Escherichia coli XL2-Blue MRF9 (Stratagene) andamplified according to the supplier’s recommendations. Immunoscreen-ing was carried out by standard methodology (Sambrook et al., 1989)using a combination of 3 sera from Peruvian patients infected with L.braziliensis. Selection of patients was carried out by observing parasiteson the cutaneous lesions and subsequently positive in vitro culture.From this screening, 5 positive clones were isolated. All clones werepartially sequenced on an ALF-express automatic sequencer (PharmaciaBiotech), according to the supplier’s descriptions. Analysis of nucleotideand aa sequences was performed using University of Wisconsin Genet-ics Computer Group programs by accessing GenBank and using EMBLand SwissProt databases of DNA (Altschul et al., 1990). LbcIV clone,which presented sequence homology to L6 ribosomal protein, was se-lected for this study, and both strands were sequenced.

Genomic DNA from L. braziliensis promastigotes was isolated ac-cording to standard procedures (Sambrook et al., 1989), digested withAvaI, PvuII, NruI, SalI, NotI, EcoRV, HindIII, HindII, BamHI, NarI,and PstI restriction endonucleases and also partially with PstI enzyme,electrophoresed in 0.8% agarose gels, and transferred to Z-probe mem-branes (BioRad, Hercules, California). Five micrograms of cytoplasmicRNA, isolated as described by Chomczinsky and Sacchi (1987), wassize fractionated on 1% agarose–formaldehyde gel and immobilized onnylon membranes. Poly(A)1 RNA fraction was purified from total RNAby oligo-d(T) cellulose chromatography according to standard proce-dures (New England Biolabs, Beverly, Massachusetts).

For pulsed-field gel electrophoresis (PFGE) analysis, agarose blockscontaining 5 3 107 parasites were prepared as described (Clark et al.,1990) and stored at 4 C in 0.5 M ethylenediaminetetraacetic acid(EDTA), pH 9.5. One-fifth of each block was electrophoresed (1% aga-rose in 0.53 Tris–borate–EDTA) at 120 V for 64 hr at 15 C with apulse time of 75–250 sec and gel transferred to nylon filters (BioRad).Hybridizations for either DNA or RNA analysis were carried out over-night at 42 C in 50% formamide (v/v), 53 standard saline citrate (SSC,13 SSC is 0.15 M NaCl–0.015 M sodium citrate, pH 7.0), 0.2% sodiumdodecyl sulfate (SDS), 53 Denhart’s, 0.01 M Na2HPO4–NaH2PO4, and0.1 mg ml21 of herring sperm DNA. After hybridization, filters weretwice washed in 2 3 SSC–0.1% SDS for 10 min at room temperatureand once at 65 C in 0.13 SSC–0.1% SDS for 30 min. A 393-nucleotidefragment (LbL6cr) corresponding to the 39 end of the L6 coding regionwas generated by polymerase chain reaction using LbcIV clone as thetemplate and primers C8s (59-CCCGATGAAGTATAGCGGCGTCCCC-39) and C8as (59-TGTGCTTACCAGTTCCAGCGGTGCG-39) as theprobe. The fragment was purified from agarose gel using Qiaex II GelExtraction Kit (Qiagen, Hilden, Germany) and random primer labeledusing [a-32P]-dCTP and Rediprime DNA labeling system (AmershamBiosciences) as recommended by the manufacturer.

Leishmania braziliensis promastigotes in logarithmic growth phasewere treated with the RNA synthesis inhibitor actinomycin D. Tran-scriptional inhibition was measured by determination of [5-3H]-uridineincorporation into RNA in treated and untreated cultures at differenttimes after addition of the drug. To estimate RNA synthesis, aliquotsof cultures treated with 15 mg ml21 of actinomycin D were taken after1, 3, 6, 9, and 12 hr and incubated in presence of 3 mCi of [5-3H]-uridine (Amersham Biosciences) at 22 C for 30 min. The 3H incorpo-ration was determined using the MultiScreen assay system (Millipore,Bedford, Massachusetts) according to the manufacturer’s instructionsafter the addition of cold 10% trichloroacetic acid. The mRNA levelswere measured after isolation of total RNA from treated and untreatedcultures and Northern blot analysis.

Five clones gave a positive hybridization signal after immunoscreen-ing of a L. braziliensis cDNA expression library using a pool of serafrom cutaneous leishmaniosis patients. Sequence analysis of one ofthese clones (named LbcIV) revealed an insert of 813 bp in length,whose sequence (accession number AF131910) is shown in Figure 1.The deduced aa sequence showed the presence of a single 579-nucle-otide open reading frame, which codes for a 192-aa protein (Fig. 1)with a theoretical molecular weight of 20.9 kDa. Nucleotide sequenceanalysis showed the presence of the miniexon-spliced leader sequence,indicating that the transsplicing acceptor signal is located 32 nucleotidesupstream from the ATG initiation codon. Moreover, a polyadenine trackof 17 nucleotide was found at the end of a 164-nucleotide-long 39-untranslated region (UTR). The aa sequence homology with sequencespresent in the SwissProt database through BLAST analysis evidenced

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FIGURE 2. A. Alignment of the L6 ribosomal deduced aa sequence from L. braziliensis (L.b.), R. norvegicus (Rat.), Homo sapiens (human),C. albicans (C.a.), and S. cerevisiae (S.c.). Black boxes indicate identical residues, and gray boxes indicate similar aa residues. Gaps are representedby dots. Numbers to the left of the sequences indicate the aa position. B. Representation of conserved domains in the deduced aa sequence ofL6 ribosomal protein from L. braziliensis, accession number AF131910 (L.b.); R. norvegicus, accession number AAH61784 (Rat.); H. sapiens,accession number AAH32299 (human); C. albicans, accession number Q9P834 (C.a.); S. cerevisiae, accession number NP013638 (S.c.); andCaenorhabditis elegans, accession number P47991 (C.el.). Domains are indicated in bold. Superscript numbers indicate the aa position of thedomains inside each sequence.

high similarities between the LbcIV deduced aa sequence and L6 ri-bosomal proteins from a wide variety of eukaryotic organisms. Thus,as shown in Figure 2A, the L. braziliensis L6 ribosomal protein shows44.8% identity with L6 ribosomal protein from Rattus norvergicus(P21533), 46.2% identity with the human L6 (Q02878), and 52.2 and46.9% identity with L6 ribosomal proteins from Saccharomyces cer-evisiae (Q02326) and Candida albicans (CAB77645.1), respectively.Four hypothetical functional motifs, 2 protein kinase C phosphorylationsites (aa 17–19 and 85–87), 1 amidation site (aa 44–47), and 1 tyrosinekinase phosphorylation site (aa 74–80) were observed in the L. brazil-iensis L6 protein, which are conserved and maintained at similar po-sitions in the homologous L6 protein from yeast (Fig. 2B). In addition,3 of these 4 domains are also present in L6 proteins from mice andhumans. Interestingly, L6 ribosomal protein from L. braziliensis has a

polypeptide of 14 aa (aa 129–142) that is not found in any L6 ribosomalproteins described to date. This specific region is surrounded by frag-ments of low homology with the described L6 proteins.

The genomic organization of the L. braziliensis L6 gene was studiedby Southern blot analysis and chromosomal blotting of L. braziliensisgenomic DNA using a 393-bp L6 coding region fragment as a probe(see Materials and Methods). Figure 3A shows the hybridization patternobtained after digestion of genomic DNA with several restriction en-zymes, including AvaI, EcoRV, HindII, and PstI, which cut once andPvuII, NruI, and NarI, which cut twice within the LbcIV clone, andSalI, NotI, HindIII, and BamHI, which do not cut into LbcIV clone.The presence of 2 bands in the lane containing DNA digested with SalI,HindIII, and BamHI, which do not cut inside L. braziliensis L6 gene,suggests the presence of more than 1 copy of the gene. Analysis of the

RESEARCH NOTES 911

FIGURE 3. Southern blot analysis of the L. braziliensis L6 ribosomal gene. A. Promastigote L. braziliensis genomic DNA (2 mg) was digestedwith AvaI, PvuII, NruI, SalI, NotI, EcoRV, HindIII, HindII, BamHI, NarI, and PstI restriction enzymes and separated by 0.8% agarose gelelectrophoresis. MW, DNA molecular weight in kilobases. B. Promastigote L. braziliensis genomic DNA (2 mg) was partially digested with PstIenzyme and separated by 0.8% agarose gel electrophoresis. After blotting, filters were hybridized with the [a-32P]-labeled LbL6cr DNA fragment.Digestion time, from 5 min to 3 hr, is indicated at the top of the figure. MW, DNA molecular weight in kilobases. The hybridization bands aremarked as a, b, c and d on the right of the panel. C. Schematic representation of L. braziliensis L6 ribosomal gene locus deduced from Southernblot analysis and hybridization. Open boxes indicate coding regions. Dotted boxes correspond to the probe used. AvaI (A), PvuII (Pv), NruI (Nr),EcoRV (E), HindII (H), NarI (N), and PstI (P) restriction enzymes. a, b, c, and d represent the fragments generated as a result of genomic DNAPstI total digestion. The size of the generated fragments by PstI digestion is indicated in kilobases above each lane.

size of the obtained hybridization bands indicates that there are 2 copiesarranged in tandem arrays and separated by an intergenic region ofapproximately 3.8 kb. Thus, EcoRV, PvuII, and NruI enzymes liberatea band of approximately 4.4 kb, a size that correspond to 1 gene unitplus the intergenic region of 2 copies organized in tandem. The hybrid-ization pattern obtained by PstI partial digestion shown in Figure 3Bsupports the existence of 2 copies in tandem arrays of the L. braziliensisL6 gene. A schema of the genomic organization of the L6 cluster isshown in Figure 3C. The pattern of bands obtained does not indicatedivergence between the coding sequences of the 2 copies. However,39UTR showed divergence for NarI enzyme. To know the chromosomallocation of ribosomal L6 gene cluster, PFGE and hybridization withLbL6cr as probe were used. As shown in Figure 4, only 1 strong signalwas seen at a position corresponding to a chromosome size of approx-imately 0.9 Mb.

The presence of the L. braziliensis L6 transcripts was determined byNorthern blot of total RNA and polyadenylated RNA fractions frompromastigotes using the L. braziliensis L6 coding region as a probe.Two hybridization bands of approximately 1.15 and 0.85 nucleotideswere detected in both lanes, indicating that the 2 copies of the L. bra-ziliensis L6 gene are transcribed as 2 polyadenylated messengers ofdifferent sizes (Fig. 5A). Densitometric analysis of the hybridizationbands revealed that the ratio of the mRNA steady-state levels betweenboth mature transcripts was 1:3 and that the smaller one was more

abundant than the larger one. The same hybridization bands were de-tected when the filter was rehybridized with a probe corresponding tothe 39UTR (data not shown).

To investigate whether differences in the relative abundance of L.braziliensis L6 ribosomal mRNAs were correlated with a different half-life of the transcripts, the abundance of L6 transcripts was analyzed inpromastigote cultures treated with the transcription inhibitor actinomy-cin D. The stability of both mRNAs was determined by Northern blotanalysis and hybridization with the LbL6cr fragment as probe. As ob-served in Figure 5B, after 6 hr of actinomycin D treatment, the abun-dance of 0.85-kb ribosomal L6 transcripts decreased to the point thatonly traces were detected. Remarkably, stability of the 0.85-kb tran-scripts was lower than that of the 1.15-kb transcripts and similar to thatof the b-tubulin transcripts (Fig. 5B). A densitometric analysis indicateda half-life of 3.5 and 6.5 hr, respectively, for the 0.85- and 1.15-kbtranscripts.

To determine the relationship between the steady-state level of theL6 mRNAs and the parasite growth phase, a Northern blot containingsimilar amounts of RNA from promastigotes in different growth phaseswas probed with the radiolabeled LbL6cr fragment. To detect any dif-ferences in the loading of RNA amounts among lanes, the filter wasrehybridized with a Trypanosoma cruzi 18S rDNA probe. As shown inFigure 5C, no variation was found in the steady-state level of L6 0.85-kb transcripts for the parasites in the logarithmic phase of growth rel-

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FIGURE 4. PFGE of L. braziliensis chromosomes. 1. Ethidium bro-mide staining of PFGE-separated chromosomes from S. cerivisiae(Pharmacia Biotech) (a) and L. braziliensis (b). 2. After blotting, chro-mosomes were hybridized with [a-32P]-labeled LbL6cr sequence. Arrowindicates the position of the chromosome that contains L6 genes.

FIGURE 5. Northern blot analysis of the L. braziliensis L6 ribosomaltranscripts. A. Five micrograms of total (T) and 0.2 mg of polyadenyl-ated (A1) RNA from L. braziliensis promastigotes were separated on1% agarose–formaldehyde gel, transferred to a nylon membrane, andhybridized with LbL6cr as the probe. The size of the hybridizing bands(kb) is indicated on the left of the panel. B. Total RNA from L. brazil-iensis promastigotes treated with actinomycin D for 0, 1, 3, 6, 9, and12 hr was size fractionated and transferred to the nylon membrane asmentioned above and hybridized with LbL6cr (LbL6), 18S rRNA (18S),and L. major b-tubulin (b-tub) [a-32P]-labeled sequences. Percentage ofinhibition of uridine incorporation is indicated at the bottom of thefigure. C. Five micrograms of total RNA from L. braziliensis promas-tigotes grown in the logarithmic growth phase, 3.5 3 106 parasites ml21

(L), and stationary growth phase, 65 3 106 parasites ml21 (S), was sizefractionated and transferred to nylon membranes as mentioned aboveand hybridized with LbL6cr (LbL6) or 18S rRNA (18S) [32P]-labeledsequences as probes.

ative to the stationary phase. However, a slight increase in the abun-dance of the L6 1.15-kb transcripts was observed when parasitesreached the stationary phase of growth.

The ribosome is a ribonucleoprotein complex that performs the cru-cial function of protein biosynthesis and plays an essential role in cellproliferation. In trypanosomatids and other lower eukaryotes, knowl-edge regarding ribosomal proteins and their coding genes is poor andfocused only on the antigenic acidic ribosomal proteins (Requena et al.,2000). Moreover, little is known about ribosomal genes in terms of theirgenomic organization and mechanism of expression.

Screening of a L. braziliensis cDNA expression library using a poolof sera from cutaneous leishmaniasis patients has permitted the isolationof different genes coding for conserved proteins (Gonzalez et al., 2002;Martinez et al., 2002). In fact, several Leishmania spp. genes codingfor evolutionarily conserved proteins (Requena et al., 2000) have re-cently been cloned using expression libraries screening with sera frominfected animals and leishmaniasis patients. The data presented in thisarticle show that the LbcIV clone highly recognized by leishmaniasispatient sera contains a single open reading frame that codes for a poly-peptide with 192 aa. BLASTA analysis indicated a protein identity thatcorresponds to the L6 ribosomal protein, which possesses the structuralcharacteristic reported for eukaryotic L6 ribosomal proteins. Four func-tional hypothetical motifs are present, 2 protein kinase C phosphory-lation sites, an amidation site, and a tyrosine kinase phosphorylationsite. These motifs are located in equivalent positions in all describedL6 ribosomal proteins, with the exception of the tyrosine kinase site,which is not present in the human, rat, and C. elegans proteins. Thehigh conservation degree of these motifs by eukaryotes could be anindication of their importance for the L6 protein functionality. Interest-ingly, the L. braziliensis L6 protein contains a specific sequence of 14aa that is absent in the other L6 proteins. Likewise, the L. braziliensisL6 protein possesses a tyrosine kinase motif that is absent in human,rat, and C. elegans L6 proteins. On the other hand, a sequence similarityof 38% was also detected between the L. braziliensis L6 ribosomalprotein and the previously described histone H1 (Martinez et al., 2002)from this parasite. A pairwise comparison of L. braziliensis ribosomalL6 and H1 histones from different organisms also gave high aa iden-tities. For instance, L. braziliensis L6 has 27.9% identity with the Ar-abidopsis thailana H1 protein for 166 aa and 28.3% identity with Homosapiens H1 protein for 66 residues. This finding has been reported pre-viously between rat L6 and H1 proteins from different species (Chanand Wool, 1996), suggesting that the sequence homology could be be-

cause of a similar protein functionality such as binding to nucleic acidsor a common evolutionary origin.

Analysis of the genomic organization of the L. braziliensis ribosomalL6 genes showed the presence in the genome of 2 genes organized intandem array, which are located in a chromosome of approximately 0.9Mb. Similar tandem organizations have also been reported for otherLeishmania spp. ribosomal genes (Soto, Requena, and Alonso, 1993;Soto, Requena, Garcia et al., 1993). The L. braziliensis L6 gene islocated in a chromosome of 0.9 Mb, which also contains the L. brazil-iensis L14 gene (Gonzalez et al., 2004). In addition, we show that 2size classes of polyadenylated mRNA, 0.85 and 1.15 kb, result probablyfrom transcription of the 2 gene units. The level of both mRNAs isclearly different although this difference is not correlated with the sta-bility of the transcripts. The data presented in this article show that theshorter messenger is more abundant than the longer one, although itsstability is clearly lower. Thus, if both genes are transcribed as a poly-cistronic precursor, a regulatory process should exist at the maturationlevel in which 39UTRs are probably implicated. In fact, the 39-untrans-lated sequences of many trypanosomatid transcripts are thought to beresponsible for its regulation (Flinn et al., 1992; Maranon et al., 2000).The biological significance of 2 mRNAs encoding the same ribosomalprotein is unknown but probably is associated with the necessary avail-ability of the protein during the life cycle. Thus, a relative increase inthe 1.15-kb mRNA-level transcripts was observed in the stationary

RESEARCH NOTES 913

phase of growth, when the division process has stopped and the differ-entiation mechanism started. Further studies to identify the putative reg-ulatory sequences are in progress.

We are grateful to Manuel C. Lopez from the encouragement andhelpful discussion. E.C. was supported by Ministerio de Educacion yCiencia Predoctoral Fellowship. This work was supported by Grant 01/145 from Servicio Andaluz de Salud, Junta de Andalucıa, Spain, GrantPIO21511 from Fondo de Investigaciones Sanitarias, Spain, and GrantC03/04 RICET, Instituto de Salud Carlos III, Spain.

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