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Jose Requena

Universidad Autónoma de Madrid, Departamento de Biología Molecular, Faculty Member

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Jose M. Requena received his doctorate in 1990 from the Department of Molecular Biology of the Autonomous University of Madrid (UAM). From 1998 to present he is Associate Professor at Universidad Autonoma de Madrid. Since 2003, he is group leader at the Severo Ochoa Molecular Biology Center (CBMSO). His research activity has been developed in the area of molecular biology and biomedicine, working mainly in aspects of genomic organization and regulation of gene expression in Leishmania, and also in diagnostic strategies and development of immunoprophylactic systems against leishmaniasis.

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Papers by Jose Requena

The heat shock proteins, Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Cell Stress & Chaperones, 2002

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins ... more Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coli maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship

BMC Genomics, 2008

Background: Protozoan parasites of the genus Leishmania are causative agents of a diverse spectru... more Background: Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. Results: By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. Conclusion: SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in posttrancriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

Lights and shadows on gene organization and regulation of gene expression in Leishmania

Frontiers in Bioscience, 2011

Introduction 3. Chromosomal organization and gene transcription 4. Copy number of genes and regul... more Introduction 3. Chromosomal organization and gene transcription 4. Copy number of genes and regulation of gene expression 5. Regulation of mRNA processing 6. Regulation of mRNA stability through cis-acting elements 7. Control of mRNA translation 8. Transacting factors and their involvement in specific regulatory mechanisms of gene expression 9. Regulation at the post-translational level 10. Conclusions and perspectives 11. Acknowledgments 12. References

Omics approaches for understanding gene expression in Leishmania: clues for tackling leishmaniasis

Protozoan Parasitism: From Omics to Prevention and Control, 2018

Editorial: Protozoan parasites in the multi-omics era: present and future

Frontiers in Cellular and Infection Microbiology, Sep 13, 2023

Editorial on the Research Topic Protozoan parasites in the multi-omics era: present and future Pa... more Editorial on the Research Topic Protozoan parasites in the multi-omics era: present and future Parasitic diseases contribute significantly to the global human disease burden. Many of them are classified into the group of Neglected Tropical Diseases (NTDs), largely due to insufficient attention from public funding agencies in developed countries. Collectively, NTDs impact over 1 billion people globally, predominantly in rural areas of low-income countries. At present, there is no vaccine against parasitic diseases of general acceptance (Solana et al., 2022). The currently available chemotherapies, overall, are poor and suffer from serious side effects, limited efficacy, and resistance development. Therefore, understanding parasites at the molecular level has become essential for designing new strategies to control these devastating diseases. In the last decade, new technological advances have forever changed the way genomes, transcriptomes, proteomes, and metabolomes are analyzed, successfully generating huge amounts of molecular data for parasites, hosts, and their interactions. Furthermore, these technologies (known as 'omic' approaches) have become increasingly accessible in terms of cost and time, promoting their implementation in a growing number of specialized research laboratories worldwide (Figure 1). Omic studies, including but not limited to genome sequencing, transcriptomics, proteomics, metabolomics, and epigenetics, are contributing to addressing specific biological questions, such as drug resistance, epidemiology, genetic exchange, immune evasion mechanisms, genome organization, and gene expression. Equally important, their application in disease surveillance, disease outcomes, and interactions with hosts and vectors will have positive impact in improving human health. Thus, this Research Topic was intended to spotlight recent advances in the field of parasite omics applied to study protozoan pathogens. It includes articles focused on parasites with a high impact in human health; four of them deal with causative agents of the so-called NTDs by the World Health Organization (WHO, 2023). These are: Trypanosoma cruzi, the causative agent of the Chagas disease, an endemic disease in Latin America but expanded globally through human migrations. Second, Giardia intestinalis, the causative of Frontiers in Cellular and Infection Microbiology frontiersin.org 01

Aportes a la evolución y clasificación taxonómica de los géneros de importancia médica de la familia Trypanosomatidae basado en el análisis filogenético del gen de la proteína de choque térmico 70

Anales de la Academia de Ciencias de Cuba, 2021

Introducción: Dentro de la familia Trypanosomatidae hay dos géneros de importancia médica por su ... more Introducción: Dentro de la familia Trypanosomatidae hay dos géneros de importancia médica por su papel como patógenos humanos, Trypanosoma y Leishmania. Dentro de estos géneros se encuentran especies y subespecies causantes de la leishmaniasis, la tripanosomiasis americana y la tripanosomiasis africana humana. Objetivos: En este estudio investigamos las relaciones filogenéticas de los géneros Leishmania y Trypanosoma utilizando la secuencia del gen hsp70 citoplasmática y discutimos las implicaciones para las definiciones de especies, la evolución y la taxonomía de estos géneros. Métodos: Se secuenció el gen de la HSP70 de 44 cepas de 11 especies de Leishmania y 41 cepas de 9 especies de Trypanosoma de diferentes orígenes geográficos. Las secuencias obtenidas se alinearon con las secuencias publicadas previamente en las bases de datos. El programa MEGA se utilizó para la construcción de distintos árboles filogenéticos basados en parámetros de distancia y caracteres. El soporte de los...

Molecular and functional characterization of adenylate kinase 2 gene from<i>Leishmania donovani</i>

European journal of biochemistry, Oct 16, 2003

Backgroud: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in pho... more Backgroud: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP + to NADPH using the electrons provided by Ferredoxin (Fd), a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd. Methods: The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5′RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking. Results: The kinetic analysis shows K M NADPH of 12.5 M and a k cat of 86 s −1 , data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS , sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS. Conclusion: The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.

Analysis of Post-transcriptional Regulation Operating on Transcription Products of the Tandemly Linked Leishmania infantum hsp70 Genes

Journal of Biological Chemistry, Feb 1, 1997

The genomic organization and expression of the hsp70 genes of Leishmania infantum were examined. ... more The genomic organization and expression of the hsp70 genes of Leishmania infantum were examined. In the cluster there are at least six copies of the hsp70 genes arranged in a head-to-tail tandem of 3.8-kilobase repetition units. The hsp70 gene copy (gene 6) located at the 3 end of the tandem has a 3-untranslated region highly divergent in sequence relative to the 3-untranslated region of the rest of hsp70 gene copies (genes 1-5). Nuclease S1 protection assays indicated that the steadystate level of the mRNAs derived from gene 6 is about 50-fold more abundant than the transcript level derived from genes 1-5. Nuclear run-on assays showed, however, that all hsp70 genes are transcribed at similar rates. Thus, it is likely that the differences in the steady-state levels of the transcripts from the hsp70 genes should be associated with variations in their processing or maturation rates. While the abundance of the mRNAs derived from hsp70 genes 1-5 is increased by heat shock, the hsp70 gene 6 mRNA level remains unaffected. Our data showed that ongoing protein synthesis is required for the maintenance of the heat inducement, depicting, thus, a post-transcriptional mechanism of positive regulation involving a labile protein factor that would be either induced or activated during heat shock.

Leishmania infantum (JPCM5) Transcriptome, Gene Models and Resources for an Active Curation of Gene Annotations

Genes

Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe for... more Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe form of leishmaniasis. An improved assembly for the L. infantum genome was published five years ago, yet delineation of its transcriptome remained to be accomplished. In this work, the transcriptome annotation was attained by a combination of both short and long RNA-seq reads. The good agreement between the results derived from both methodologies confirmed that transcript assembly based on Illumina RNA-seq and further delimitation according to the positions of spliced leader (SAS) and poly-A (PAS) addition sites is an adequate strategy to annotate the transcriptomes of Leishmania, a procedure previously used for transcriptome annotation in other Leishmania species and related trypanosomatids. These analyses also confirmed that the Leishmania transcripts boundaries are relatively slippery, showing extensive heterogeneity at the 5′- and 3′-ends. However, the use of RNA-seq reads derived from ...

From Omics to Prevention and Control

Leishmaniases, a group of parasitic diseases caused by species of the genus Leishmania, a"ic... more Leishmaniases, a group of parasitic diseases caused by species of the genus Leishmania, a"ict millions of people across the globe and cause signi#cant morbidity and mortality. Unfortunately, vaccine and chemotherapy options are limited. $e advances in whole genome sequencing have led to renewed impetus in identifying druggable targets for future development of more e%ective treatments. Hence, the last decade has witnessed a revolution in our understanding of the Leishmania genomes through the completion of an increasing number of genome sequencing projects for several species and strains. However, the completion of a genome sequence is not the #nal product, rather it is just the beginning towards the objective of linking the wealth of data encoded in millions of bases to the biological processes of an organism. Moreover, the genome features (genomics) is only a part of the problem to be solved: the genome yields on transcription, the transcriptome, which in turn yields the prot...

ARAMIS: From systematic errors of NGS long reads to accurate assemblies

Briefings in Bioinformatics, 2021

NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio)... more NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio) have revolutionized the sequencing field over the last decade improving multiple genomic applications like de novo genome assemblies. However, their error rate, mostly involving insertions and deletions (indels), is currently an important concern that requires special attention to be solved. Multiple algorithms are available to fix these sequencing errors using short reads (such as Illumina), although they require long processing times and some errors may persist. Here, we present Accurate long-Reads Assembly correction Method for Indel errorS (ARAMIS), the first NGS long-reads indels correction pipeline that combines several correction software in just one step using accurate short reads. As a proof OF concept, six organisms were selected based on their different GC content, size and genome complexity, and their PacBio-assembled genomes were corrected thoroughly by this pipeline. We fo...

Identificación de proteínas reguladoras de la expresión génica en tripanosomátidos

Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales, 2018

Los tripanosomátidos son parásitos causantes de patologías de reconocido impacto en salud pública... more Los tripanosomátidos son parásitos causantes de patologías de reconocido impacto en salud pública como la enfermedad de Chagas, la enfermedad del sueño y la leishmaniasis. Estos microorganismos divergieron tempranamente de la línea evolutiva de los eucariotas y se caracterizan por poseer mecanismos peculiares de regulación génica finamente orquestados, tan eficaces que han asegurado su transmisión al permitirles adaptarse a ambientes inhóspitos y dispares como los de sus huéspedes invertebrados y mamíferos. Como consecuencia de su peculiar organización genómica, los tripanosomátidos han apostado por regular la expresión de sus genes a través de mecanismos posteriores a la transcripción, mediados principalmente por la acción de proteínas de unión a ARN (RNA-binding proteins, RBP), que reconocen su mensajero blanco gracias a la presencia de elementos reguladores en cis y se asocian con el ARN formando complejos ribonucleoprotéicos. De esta manera, las células establecen redes regula...

Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing

Universitas Scientiarum, 2018

In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mec... more In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of them RNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYT1 and kLYT1, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5’ and 3’ UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5’ UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T.cruzi DTU. In additi...

Analysis of the Antigenic and Prophylactic Properties of the Leishmania Translation Initiation Factors eIF2 and eIF2B in Natural and Experimental Leishmaniasis

Frontiers in Cellular and Infection Microbiology, Apr 5, 2018

Different members of intracellular protein families are recognized by the immune system of the ve... more Different members of intracellular protein families are recognized by the immune system of the vertebrate host infected by parasites of the genus Leishmania. Here, we have analyzed the antigenic and immunogenic properties of the Leishmania eIF2 and eIF2B translation initiation factors. An in silico search in Leishmania infantum sequence databases allowed the identification of the genes encoding the α, β, and γ subunits and the α, β, and δ subunits of the putative Leishmania orthologs of the eukaryotic initiation factors F2 (LieIF2) or F2B (LieIF2B), respectively. The antigenicity of these factors was analyzed by ELISA using recombinant versions of the different subunits. Antibodies against the different LieIF2 and LieIF2B subunits were found in the sera from human and canine visceral leishmaniasis patients, and also in the sera from hamsters experimentally infected with L. infantum. In L. infantum (BALB/c) and Leishmania major (BALB/c or C57BL/6) challenged mice, a moderate humoral response against these protein factors was detected. Remarkably, these proteins elicited an IL-10 production by splenocytes derived from infected mice independently of the Leishmania species employed for experimental challenge. When DNA vaccines based on the expression of the LieIF2 or LieIF2B subunit encoding genes were administered in mice, an antigen-specific secretion of IFN-γ and IL-10 cytokines was observed. Furthermore, a partial protection against murine CL development due to L. major infection was generated in the vaccinated mice. Also, in this work we show that the LieIF2α subunit and the LieIF2Bβ and δ subunits have the capacity to stimulate IL-10 secretion by spleen cells from naïve mice. B-lymphocytes were identified as the major producers of this anti-inflammatory Garde et al. Leishmania Antigenic eIF2 and eIF2B cytokine. Taking into account the data found in this study, it may be hypothesized that these proteins act as virulence factors implicated in the induction of humoral responses as well as in the production of the down-regulatory IL-10 cytokine, favoring a pathological outcome. Therefore, these proteins might be considered markers of disease.

The <i>Leishmania infantum</i> acidic ribosomal protein LiP2a induces a prominent humoral response <i>in vivo</i> and stimulates cell proliferation <i>in vitro</i> and interferon-gamma (IFN-<i>γ</i>) production by murine splenocytes

Clinical and Experimental Immunology, Nov 1, 2000

The acidic ribosomal proteins of the protozoan parasite Leishmania infantum have been described a... more The acidic ribosomal proteins of the protozoan parasite Leishmania infantum have been described as prominent antigens during both human and canine visceral leishmaniasis. In this study we present data showing that the intraperitoneal administration in BALB/c mice of the Leishmania LiP2a protein, in the absence of any added adjuvants, elicited a strong humoral response as an indication that the protein is a potent immunogen. Despite the evolutionary conservation of the acidic ribosomal proteins, the antibody response was found to be specific for the Leishmania protein. Another remarkable finding was the observation that the LiP2a protein stimulates the in vitro proliferation of splenocytes from either LiP2a-immunized or naive BALB/c mice. Since similar proliferative indices were observed in T cell-enriched cultures, it is likely that the LiP2a stimulating activity is due mainly to T lymphocyte expansion. Also, the stimulatory effect was demonstrated to be antigen-specific, since the proliferation was abrogated by the presence of anti-LiP2a antibodies. Interestingly, the LiP2a protein stimulated the production of substantial amounts of IFN-γ in cultured splenocytes from LiP2a-immunized mice. Our data indicate therefore that the immunostimulatory properties shown by this antigen should be taken into account when developing therapeutic and prophylactic vaccines against leishmaniasis.

The <i>Leishmania infantum</i> Acidic Ribosomal Protein P0 Administered as a DNA Vaccine Confers Protective Immunity to <i>Leishmania major</i> Infection in BALB/c Mice

Infection and Immunity, Nov 1, 2003

In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal... more In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-␥) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4 ؉ and CD8 ؉ T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-␥. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.

Identification of the Leishmania infantum P0 ribosomal protein epitope in canine visceral leishmaniasis

Immunology Letters, Nov 1, 1995

In the present work we show that a high percentage of the sera from dogs naturally affected with ... more In the present work we show that a high percentage of the sera from dogs naturally affected with viscero-cutaneous leishmaniasis contain antibodies reacting with the Leishmania infantum P0 ribosomal protein. In order to map the antigenic determinants of the LiP0 protein during Leishmania-infection, the complete amino acid sequence of the protein was synthesized as overlapping 20-mer peptides. We have identified the sequence AAKEEPEESDEDDFGMG, located adjacent to the C-terminal end of the protein, as the major antigenic determinant. The anti-LiP0 antibodies present in the sera of the infected dogs do not cross-react with a relatively similar antigenic determinant of the LiP2 acidic proteins as an indication that the Leishmania P0 protein is an independent immunogenically functional antigen in the canine form of the infection.

The mRNA coding for the nucleosomal protein H2A ofLeishmaniais polyadenylated and has stem-loops at the 3′ end

Nucleic Acids Research, 1991

Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool for vaccination

Immunology Letters, Mar 1, 2001

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fuse... more The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

Organization, transcription and regulation of the <i>Leishmania infantum</i> histone H3 genes

Biochemical Journal, Sep 15, 1996

The genomic organization and transcription of the genes encoding the histone H3 of the protozoan ... more The genomic organization and transcription of the genes encoding the histone H3 of the protozoan parasite Leishmania infantum have been studied. It was found that there are multiple copies of the histone H3 genes distributed in chromosomal bands XIX and XIV. The nucleotide sequence of two of the L. infantum H3 genes, each one located in a different chromosome, is reported. Although the nucleotide sequence of the coding region of both genes is identical, the sequence of the 3h untranslated region is highly divergent. It was found also that there exist two different size classes of histone H3 transcripts, each one derived from a different gene, and that they are polyadenylated. The steady-* To whom correspondence should be addressed. The nucleotide sequence data reported in this paper appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X77591 and X95484. Abbreviations used : PFGE, pulsed-field gel electrophoresis ; UTR, untranslated region.

The heat shock proteins, Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Cell Stress & Chaperones, 2002

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins ... more Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coli maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship

BMC Genomics, 2008

Background: Protozoan parasites of the genus Leishmania are causative agents of a diverse spectru... more Background: Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. Results: By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. Conclusion: SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in posttrancriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

Lights and shadows on gene organization and regulation of gene expression in Leishmania

Frontiers in Bioscience, 2011

Introduction 3. Chromosomal organization and gene transcription 4. Copy number of genes and regul... more Introduction 3. Chromosomal organization and gene transcription 4. Copy number of genes and regulation of gene expression 5. Regulation of mRNA processing 6. Regulation of mRNA stability through cis-acting elements 7. Control of mRNA translation 8. Transacting factors and their involvement in specific regulatory mechanisms of gene expression 9. Regulation at the post-translational level 10. Conclusions and perspectives 11. Acknowledgments 12. References

Omics approaches for understanding gene expression in Leishmania: clues for tackling leishmaniasis

Protozoan Parasitism: From Omics to Prevention and Control, 2018

Editorial: Protozoan parasites in the multi-omics era: present and future

Frontiers in Cellular and Infection Microbiology, Sep 13, 2023

Editorial on the Research Topic Protozoan parasites in the multi-omics era: present and future Pa... more Editorial on the Research Topic Protozoan parasites in the multi-omics era: present and future Parasitic diseases contribute significantly to the global human disease burden. Many of them are classified into the group of Neglected Tropical Diseases (NTDs), largely due to insufficient attention from public funding agencies in developed countries. Collectively, NTDs impact over 1 billion people globally, predominantly in rural areas of low-income countries. At present, there is no vaccine against parasitic diseases of general acceptance (Solana et al., 2022). The currently available chemotherapies, overall, are poor and suffer from serious side effects, limited efficacy, and resistance development. Therefore, understanding parasites at the molecular level has become essential for designing new strategies to control these devastating diseases. In the last decade, new technological advances have forever changed the way genomes, transcriptomes, proteomes, and metabolomes are analyzed, successfully generating huge amounts of molecular data for parasites, hosts, and their interactions. Furthermore, these technologies (known as 'omic' approaches) have become increasingly accessible in terms of cost and time, promoting their implementation in a growing number of specialized research laboratories worldwide (Figure 1). Omic studies, including but not limited to genome sequencing, transcriptomics, proteomics, metabolomics, and epigenetics, are contributing to addressing specific biological questions, such as drug resistance, epidemiology, genetic exchange, immune evasion mechanisms, genome organization, and gene expression. Equally important, their application in disease surveillance, disease outcomes, and interactions with hosts and vectors will have positive impact in improving human health. Thus, this Research Topic was intended to spotlight recent advances in the field of parasite omics applied to study protozoan pathogens. It includes articles focused on parasites with a high impact in human health; four of them deal with causative agents of the so-called NTDs by the World Health Organization (WHO, 2023). These are: Trypanosoma cruzi, the causative agent of the Chagas disease, an endemic disease in Latin America but expanded globally through human migrations. Second, Giardia intestinalis, the causative of Frontiers in Cellular and Infection Microbiology frontiersin.org 01

Aportes a la evolución y clasificación taxonómica de los géneros de importancia médica de la familia Trypanosomatidae basado en el análisis filogenético del gen de la proteína de choque térmico 70

Anales de la Academia de Ciencias de Cuba, 2021

Introducción: Dentro de la familia Trypanosomatidae hay dos géneros de importancia médica por su ... more Introducción: Dentro de la familia Trypanosomatidae hay dos géneros de importancia médica por su papel como patógenos humanos, Trypanosoma y Leishmania. Dentro de estos géneros se encuentran especies y subespecies causantes de la leishmaniasis, la tripanosomiasis americana y la tripanosomiasis africana humana. Objetivos: En este estudio investigamos las relaciones filogenéticas de los géneros Leishmania y Trypanosoma utilizando la secuencia del gen hsp70 citoplasmática y discutimos las implicaciones para las definiciones de especies, la evolución y la taxonomía de estos géneros. Métodos: Se secuenció el gen de la HSP70 de 44 cepas de 11 especies de Leishmania y 41 cepas de 9 especies de Trypanosoma de diferentes orígenes geográficos. Las secuencias obtenidas se alinearon con las secuencias publicadas previamente en las bases de datos. El programa MEGA se utilizó para la construcción de distintos árboles filogenéticos basados en parámetros de distancia y caracteres. El soporte de los...

Molecular and functional characterization of adenylate kinase 2 gene from<i>Leishmania donovani</i>

European journal of biochemistry, Oct 16, 2003

Backgroud: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in pho... more Backgroud: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP + to NADPH using the electrons provided by Ferredoxin (Fd), a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd. Methods: The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5′RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking. Results: The kinetic analysis shows K M NADPH of 12.5 M and a k cat of 86 s −1 , data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS , sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS. Conclusion: The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.

Analysis of Post-transcriptional Regulation Operating on Transcription Products of the Tandemly Linked Leishmania infantum hsp70 Genes

Journal of Biological Chemistry, Feb 1, 1997

The genomic organization and expression of the hsp70 genes of Leishmania infantum were examined. ... more The genomic organization and expression of the hsp70 genes of Leishmania infantum were examined. In the cluster there are at least six copies of the hsp70 genes arranged in a head-to-tail tandem of 3.8-kilobase repetition units. The hsp70 gene copy (gene 6) located at the 3 end of the tandem has a 3-untranslated region highly divergent in sequence relative to the 3-untranslated region of the rest of hsp70 gene copies (genes 1-5). Nuclease S1 protection assays indicated that the steadystate level of the mRNAs derived from gene 6 is about 50-fold more abundant than the transcript level derived from genes 1-5. Nuclear run-on assays showed, however, that all hsp70 genes are transcribed at similar rates. Thus, it is likely that the differences in the steady-state levels of the transcripts from the hsp70 genes should be associated with variations in their processing or maturation rates. While the abundance of the mRNAs derived from hsp70 genes 1-5 is increased by heat shock, the hsp70 gene 6 mRNA level remains unaffected. Our data showed that ongoing protein synthesis is required for the maintenance of the heat inducement, depicting, thus, a post-transcriptional mechanism of positive regulation involving a labile protein factor that would be either induced or activated during heat shock.

Leishmania infantum (JPCM5) Transcriptome, Gene Models and Resources for an Active Curation of Gene Annotations

Genes

Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe for... more Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe form of leishmaniasis. An improved assembly for the L. infantum genome was published five years ago, yet delineation of its transcriptome remained to be accomplished. In this work, the transcriptome annotation was attained by a combination of both short and long RNA-seq reads. The good agreement between the results derived from both methodologies confirmed that transcript assembly based on Illumina RNA-seq and further delimitation according to the positions of spliced leader (SAS) and poly-A (PAS) addition sites is an adequate strategy to annotate the transcriptomes of Leishmania, a procedure previously used for transcriptome annotation in other Leishmania species and related trypanosomatids. These analyses also confirmed that the Leishmania transcripts boundaries are relatively slippery, showing extensive heterogeneity at the 5′- and 3′-ends. However, the use of RNA-seq reads derived from ...

From Omics to Prevention and Control

Leishmaniases, a group of parasitic diseases caused by species of the genus Leishmania, a"ic... more Leishmaniases, a group of parasitic diseases caused by species of the genus Leishmania, a"ict millions of people across the globe and cause signi#cant morbidity and mortality. Unfortunately, vaccine and chemotherapy options are limited. $e advances in whole genome sequencing have led to renewed impetus in identifying druggable targets for future development of more e%ective treatments. Hence, the last decade has witnessed a revolution in our understanding of the Leishmania genomes through the completion of an increasing number of genome sequencing projects for several species and strains. However, the completion of a genome sequence is not the #nal product, rather it is just the beginning towards the objective of linking the wealth of data encoded in millions of bases to the biological processes of an organism. Moreover, the genome features (genomics) is only a part of the problem to be solved: the genome yields on transcription, the transcriptome, which in turn yields the prot...

ARAMIS: From systematic errors of NGS long reads to accurate assemblies

Briefings in Bioinformatics, 2021

NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio)... more NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio) have revolutionized the sequencing field over the last decade improving multiple genomic applications like de novo genome assemblies. However, their error rate, mostly involving insertions and deletions (indels), is currently an important concern that requires special attention to be solved. Multiple algorithms are available to fix these sequencing errors using short reads (such as Illumina), although they require long processing times and some errors may persist. Here, we present Accurate long-Reads Assembly correction Method for Indel errorS (ARAMIS), the first NGS long-reads indels correction pipeline that combines several correction software in just one step using accurate short reads. As a proof OF concept, six organisms were selected based on their different GC content, size and genome complexity, and their PacBio-assembled genomes were corrected thoroughly by this pipeline. We fo...

Identificación de proteínas reguladoras de la expresión génica en tripanosomátidos

Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales, 2018

Los tripanosomátidos son parásitos causantes de patologías de reconocido impacto en salud pública... more Los tripanosomátidos son parásitos causantes de patologías de reconocido impacto en salud pública como la enfermedad de Chagas, la enfermedad del sueño y la leishmaniasis. Estos microorganismos divergieron tempranamente de la línea evolutiva de los eucariotas y se caracterizan por poseer mecanismos peculiares de regulación génica finamente orquestados, tan eficaces que han asegurado su transmisión al permitirles adaptarse a ambientes inhóspitos y dispares como los de sus huéspedes invertebrados y mamíferos. Como consecuencia de su peculiar organización genómica, los tripanosomátidos han apostado por regular la expresión de sus genes a través de mecanismos posteriores a la transcripción, mediados principalmente por la acción de proteínas de unión a ARN (RNA-binding proteins, RBP), que reconocen su mensajero blanco gracias a la presencia de elementos reguladores en cis y se asocian con el ARN formando complejos ribonucleoprotéicos. De esta manera, las células establecen redes regula...

Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing

Universitas Scientiarum, 2018

In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mec... more In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of them RNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYT1 and kLYT1, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5’ and 3’ UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5’ UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T.cruzi DTU. In additi...

Analysis of the Antigenic and Prophylactic Properties of the Leishmania Translation Initiation Factors eIF2 and eIF2B in Natural and Experimental Leishmaniasis

Frontiers in Cellular and Infection Microbiology, Apr 5, 2018

Different members of intracellular protein families are recognized by the immune system of the ve... more Different members of intracellular protein families are recognized by the immune system of the vertebrate host infected by parasites of the genus Leishmania. Here, we have analyzed the antigenic and immunogenic properties of the Leishmania eIF2 and eIF2B translation initiation factors. An in silico search in Leishmania infantum sequence databases allowed the identification of the genes encoding the α, β, and γ subunits and the α, β, and δ subunits of the putative Leishmania orthologs of the eukaryotic initiation factors F2 (LieIF2) or F2B (LieIF2B), respectively. The antigenicity of these factors was analyzed by ELISA using recombinant versions of the different subunits. Antibodies against the different LieIF2 and LieIF2B subunits were found in the sera from human and canine visceral leishmaniasis patients, and also in the sera from hamsters experimentally infected with L. infantum. In L. infantum (BALB/c) and Leishmania major (BALB/c or C57BL/6) challenged mice, a moderate humoral response against these protein factors was detected. Remarkably, these proteins elicited an IL-10 production by splenocytes derived from infected mice independently of the Leishmania species employed for experimental challenge. When DNA vaccines based on the expression of the LieIF2 or LieIF2B subunit encoding genes were administered in mice, an antigen-specific secretion of IFN-γ and IL-10 cytokines was observed. Furthermore, a partial protection against murine CL development due to L. major infection was generated in the vaccinated mice. Also, in this work we show that the LieIF2α subunit and the LieIF2Bβ and δ subunits have the capacity to stimulate IL-10 secretion by spleen cells from naïve mice. B-lymphocytes were identified as the major producers of this anti-inflammatory Garde et al. Leishmania Antigenic eIF2 and eIF2B cytokine. Taking into account the data found in this study, it may be hypothesized that these proteins act as virulence factors implicated in the induction of humoral responses as well as in the production of the down-regulatory IL-10 cytokine, favoring a pathological outcome. Therefore, these proteins might be considered markers of disease.

The <i>Leishmania infantum</i> acidic ribosomal protein LiP2a induces a prominent humoral response <i>in vivo</i> and stimulates cell proliferation <i>in vitro</i> and interferon-gamma (IFN-<i>γ</i>) production by murine splenocytes

Clinical and Experimental Immunology, Nov 1, 2000

The acidic ribosomal proteins of the protozoan parasite Leishmania infantum have been described a... more The acidic ribosomal proteins of the protozoan parasite Leishmania infantum have been described as prominent antigens during both human and canine visceral leishmaniasis. In this study we present data showing that the intraperitoneal administration in BALB/c mice of the Leishmania LiP2a protein, in the absence of any added adjuvants, elicited a strong humoral response as an indication that the protein is a potent immunogen. Despite the evolutionary conservation of the acidic ribosomal proteins, the antibody response was found to be specific for the Leishmania protein. Another remarkable finding was the observation that the LiP2a protein stimulates the in vitro proliferation of splenocytes from either LiP2a-immunized or naive BALB/c mice. Since similar proliferative indices were observed in T cell-enriched cultures, it is likely that the LiP2a stimulating activity is due mainly to T lymphocyte expansion. Also, the stimulatory effect was demonstrated to be antigen-specific, since the proliferation was abrogated by the presence of anti-LiP2a antibodies. Interestingly, the LiP2a protein stimulated the production of substantial amounts of IFN-γ in cultured splenocytes from LiP2a-immunized mice. Our data indicate therefore that the immunostimulatory properties shown by this antigen should be taken into account when developing therapeutic and prophylactic vaccines against leishmaniasis.

The <i>Leishmania infantum</i> Acidic Ribosomal Protein P0 Administered as a DNA Vaccine Confers Protective Immunity to <i>Leishmania major</i> Infection in BALB/c Mice

Infection and Immunity, Nov 1, 2003

In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal... more In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-␥) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4 ؉ and CD8 ؉ T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-␥. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.

Identification of the Leishmania infantum P0 ribosomal protein epitope in canine visceral leishmaniasis

Immunology Letters, Nov 1, 1995

In the present work we show that a high percentage of the sera from dogs naturally affected with ... more In the present work we show that a high percentage of the sera from dogs naturally affected with viscero-cutaneous leishmaniasis contain antibodies reacting with the Leishmania infantum P0 ribosomal protein. In order to map the antigenic determinants of the LiP0 protein during Leishmania-infection, the complete amino acid sequence of the protein was synthesized as overlapping 20-mer peptides. We have identified the sequence AAKEEPEESDEDDFGMG, located adjacent to the C-terminal end of the protein, as the major antigenic determinant. The anti-LiP0 antibodies present in the sera of the infected dogs do not cross-react with a relatively similar antigenic determinant of the LiP2 acidic proteins as an indication that the Leishmania P0 protein is an independent immunogenically functional antigen in the canine form of the infection.

The mRNA coding for the nucleosomal protein H2A ofLeishmaniais polyadenylated and has stem-loops at the 3′ end

Nucleic Acids Research, 1991

Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool for vaccination

Immunology Letters, Mar 1, 2001

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fuse... more The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

Organization, transcription and regulation of the <i>Leishmania infantum</i> histone H3 genes

Biochemical Journal, Sep 15, 1996

The genomic organization and transcription of the genes encoding the histone H3 of the protozoan ... more The genomic organization and transcription of the genes encoding the histone H3 of the protozoan parasite Leishmania infantum have been studied. It was found that there are multiple copies of the histone H3 genes distributed in chromosomal bands XIX and XIV. The nucleotide sequence of two of the L. infantum H3 genes, each one located in a different chromosome, is reported. Although the nucleotide sequence of the coding region of both genes is identical, the sequence of the 3h untranslated region is highly divergent. It was found also that there exist two different size classes of histone H3 transcripts, each one derived from a different gene, and that they are polyadenylated. The steady-* To whom correspondence should be addressed. The nucleotide sequence data reported in this paper appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X77591 and X95484. Abbreviations used : PFGE, pulsed-field gel electrophoresis ; UTR, untranslated region.