The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship
© Requena et al; licensee BioMed Central Ltd. 2008
Received: 20 February 2008
Accepted: 02 June 2008
Published: 02 June 2008
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.
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.
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 post-trancriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.
Repetitive DNA sequences constitute a substantial proportion of eukaryotic genomes. For example, in mammals they account for nearly half of the genome, and in some plants they constitute up to 90% of the genome . Most of these repeated DNAs are, or were originated from, transposable elements (TEs, also known mobile elements) through transposing and duplicating events. On the basis of mechanisms of their transposition, TEs can be divided into two classes: retrotransposons, which proliferate via reverse transcription, and DNA transposons, which move strictly through DNA intermediates. Frequently, genomes harbour few active TEs; instead, genomes contains multiple repetitive elements representing remnants (or dead elements) derived from TEs . Although repetitive DNA elements have been often considered as "selfish" or "parasitic" DNAs, the now growing evidence is that these elements are involved in shaping genomes and are playing important role in epigenetic regulation of genome expression [1, 3].
Protozoan parasites of the genus Leishmania are causative agents of a complex of diseases known as leishmaniasis. The burden associated with these diseases remains important: 1.5–2 million new cases per year and 350 million people at risk in 88 countries . Apart from its impact in human health, Leishmania parasites and related trypanosomes (i.e. Trypanosoma cruzi and Trypanosoma brucei) are being extensively studied because of peculiar molecular and cellular characteristics. The genome of Leishmania major was sequenced , and more recently the genome sequences for two other Leishmania species (Leishmania infantum and Leishmania braziliensis) have been also deciphered . The comparison of these sequences reveals marked conservation of the genome architecture within the Leishmania genus, showing similar gene content and a remarkable degree of synteny . The organization of protein-coding genes into long, strand-specific, polycistronic clusters is a conspicuous feature of the Leishmania species, also observed in the T. brucei and T. cruzi genomes . This peculiar gene organization seems to be related to the lack of transcriptional control by RNA polymerase II promoters; rather, transcription initiation appears to begin in a low fidelity manner transcribing long polycistronic precursor transcripts . Despite having diverged 200 to 500 million years ago, the genomes of L. major, T. brucei and T. cruzi are highly synthenic. For example, 68 and 75% of the genes in T. brucei and L. major remain in the same gene order . In spite of this conservation in chromosome organization, the genomes of these trypanosomes differ in the content of repeated sequences. Unlike Leishmania, the genomes of T. brucei and T. cruzi are riddled with interspersed elements [10–12].
The Leishmania genome is relatively poor in repeated sequences. The first repetitive DNA sequence characterized in Leishmania corresponded to the telomeric repeats . Afterwards, multiple tandem repeats of a 60-bp sequence, named Lmet2, were found on at least six chromosomes of parasites of the L. donovani complex, being absent from other Leishmania species . Piarroux et al  characterized a low copy, repetitive DNA sequence from L. infantum that was located exclusively at a large chromosome; this sequence was detected in many other Leishmania species. A repeated sequence with features of minisatellite DNA was characterized in the L. infantum genome; this element, called LiSTIR1, is 81-bp long and G+C rich and it was found interspersed at the subtelomeric regions of four chromosomes . A 348-bp long element, designated LiR3, was found tandemly repeated within the non-transcribed spacers of the rDNA locus of L. infantum . Conserved repeats, named LCTAS, have been characterized to be adjacent to telomeres in L. braziliensis, L. major, L. mexicana and L. lainsoni . Also, several subtelomeric repetitive sequences have been characterized, showing to be responsible for size differences among the three L. major homologues for chromosome 1 . Similar repeats have been found as tandemly arranged clusters at subtelomeric regions in chromosomes 1, 19 and 22 of L. infantum. Interestingly, these repeats are transcribed by RNA polymerase II into noncoding RNAs in a developmentally regulated manner . Non-LTR retrotransposons are abundant in the genome of T. brucei and T. cruzi; by contrast, retroelements are absent from the L. major genome, where only remnants of degenerated ingi/L1Tc-related elements (or DIREs) are detectable (the L. major haploid genome contains 52 DIREs). Evolutionary analyses indicate that the trypanosomatid ancestor contained active transposable elements that have been retained in the genus Trypanosoma, but were lost in the L. major evolutionary line . Recently, in an outstanding work, Bringaud et al  have found that the L. major contains two classes of short interspersed repeated sequences, SIDER1 (785 copies) and SIDER2 (1073 copies), which displays hallmarks of trypanosomatid retroposons. Members of the SIDER1 family show high sequence similarity with a conserved 450–550-bp element, located in the 3'UTR of several Leishmania amastigote-specific transcripts, that is implicated in stage-specific translational control [23, 24]. SIDER2 elements, also located predominantly within 3'UTRs, have a demonstrated role in mRNA degradation . Thus, it was postulated that Leishmania have recycled the retroposon remnants to regulatory sequences to globally modulate the expression of a number of genes .
In the course of studying repetitive DNA in the L. infantum genome, we identified and characterized a family of repeated sequences, which are interspersed along the different chromosomes. These sequence elements are present in different Leishmania species and, here, we show a detailed analysis of these elements in the L. infantum chromosomes 20 and 32, and in the L. braziliensis and L. major chromosome 32. During the preparation of this manuscript, the existence of this class of sequences in the L. major genome was reported , and, consequently, we adopted the proposed name (SIDER, Short Interspersed Degenerated Retroposon) for the elements identified in this work.
Identification of a new family of repeated sequences in L. infantum
SIDER2 elements present in the L. infantum chromosome 32 and comparison with their homologues in L. major
L. infantum SIDER2 element
L. major SIDER2 homologue
Sequence identity (%)
SIDER elements present in the L. infantum chromosome 20
As deduced from BLAST analyses (data not shown), the rest of L. infantum chromosomes must be also populated by LiSIDER2 elements showing similar features as those described in chromosomes 20 and 32. Taking into account both the chromosomal size and the number of SIDER2s found in L. infantum chromosomes 20 and 32, we estimated that the L. infantum haploid content of SIDER2s would be around 1150 copies. This estimation is in agreement with the determination of 1073 copies of LmjSIDER2 in the L. major genome .
Sequences homologous to LiSIDER2s are also present in the genome of other Leishmania species
Since the complete sequence of the L. major is known , we carried out the same bioinformatics analysis on the L. major database using as query sequences the different LiSIDER2 elements found in the L. infantum chromosome 32 (Table 1). In all cases, the best scores were observed with sequences located in the L. major chromosome 32. Table 1 summarizes molecular features of the SIDER2s found in the L. major chromosome 32. Remarkably, it was observed an extremely high conservation, both in sequence and genomic location, of the SIDER2s found in the L. major and L. infantum chromosomes 32. To avoid confusion, following the genetic nomenclature directions for kinetoplastids , we named the L. major elements as LmjSIDER2. In an independent study, Bringaud et al  identified 55 SIDER2s elements in the L. major chromosome. Except for small variations in the coordinates, there was a total correspondence between the 54 elements identified by us (Table 1) and those identified by Bringaud and colleagues. Our analysis failed to find the LmjSIDER2 starting at position 626445 .
SIDER elements present in the L. braziliensis chromosome 32
In addition to the analysis of Leishmania genome databases, we performed searches looking for SIDER2 homologue elements in general databases (EMBL and GenBank). A large number of entries were retrieved; however, all entries contained Leishmania sequences and homologous sequences were not found in other organisms, with an intriguing exception. Thus, we found a significant homology between LiSIDER-32-121058d and the EMBL entry with accession number AM094505, which corresponds to a Lutzomyia longipalpis EST clone NSFM-162h01. Remarkably, this sandfly species acts as Leishmania transmission vector. On the other hand, BLAST searches in the T. cruzi and T. brucei genome databases (GeneDB) yielded not results, indicating that these elements are specific for the Leishmania genus. Among the retrieved entries from the EMBL and GenBank databases, there are sequences derived from L. amazonensis (U70540, AB029444, AY427440S3, DQ092336), L. braziliensis (DQ092335), L. donovani (Z94053, AC093553, AF067495, AF109296, AY028171, AY791850, DQ092337), L. hoogstraali (DQ092338), L. infantum (M93416, L27052, AJ628942, AM118098), L. major (Z54138, AY227807, AY328521, AY491007), L. mexicana (Z46971, AF350492, AJ131960, AJ427448, AJ548776, AY170465), and L. tarentolae (AY842846). Remarkably, there exist many entries corresponding to L. chagasi cDNAs (CV669830, CV667316, CV670663, CV663048, CV669851, CV669636, CV662260, CV666468, CV669797, CV666868, CV664167, CV669564, CV665051, CV663324, CV668078, CV668316) that have significant BLAST scores with SIDER2 sequences.
In a recent work, Bringaud and co-workers  identified two related families of small elements by a bioinformatics analysis of the L. major genome sequence using as bait the "79-bp signature" common to trypanosomatid retroposons . These families, named LmSIDER1 and LmSIDER2, contain 785 and 1073 copies per haploid genome, respectively. These authors raised a compelling hypothesis: these elements are extinct retroposons that have been recycled to accomplish regulatory functions for gene expression in Leishmania. Here, we describe the existence of this class of elements in the genome of L. infantum and other Leishmania species. The starting point of our work was the isolation from a macroarray of a clone showing strong hybridization signal when L. infantum total DNA was used as probe. Sequencing of this clone indicated that it contains a genomic fragment of chromosome 32, but the bioinformatics analyses showed also that this clone would contain a repeated sequence because significant homology with different sequences located on the different L. infantum chromosomes was observed. After a thoughtful analysis, we identified a total of 54 elements in the L. infantum chromosome 32 and 27 elements in the chromosome 20. Sequence comparisons analysis between the repeated elements identified in this work with those described by Bringaud and co-workers in L. major, suggest that the elements described here belong to the SIDER2 family .
SIDER2 elements show outstanding features regarding genomic organization (; this work): i) the elements are abundant and distributed along the different chromosomes in all Leishmania species; ii) the elements constitutes subfamilies related in sequence and genomic vicinity; iii) the L. major and L. infantum SIDER2s are highly conserved both in sequence and chromosomal location. This degree of conservation in chromosomal location is not maintained between the L. major/L. infantum and L. braziliensis SIDER2s (at least for chromosome 32). It should be kept in mind that L. braziliensis is the most genetically and biologically divergent of the three species analyzed for this study . A remarkable difference, which may be related with the variations in genomic distribution of SIDER2 elements among the Leishmania species, is that L. braziliensis possesses potentially active retrotransposons that are absent in the other two Leishmania species .
Accumulating data from different organisms do indicate that mobile elements and non-coding repetitive sequences are important elements in a genome and may be playing functional roles that vary from control of gene expression to chromosomal organization [1, 3]. In this regard, the sequence features and genomic organization of SIDER2 elements are suggestive of relevant functional roles, but what kind of function can they be playing? The search for these elements within coding regions in L. infantum predicted genes indicates that no SIDER2 sequences are in coding region. The sole exception to this rule is the L. infantum database entry LinJ10_V3.1340, which contains sequence homology to SIDER2s. However, this entry is considered as pseudogene, since its sequence contains several in-frame stop codons. Remarkably, this putative pseudogene shows high sequence conservation with genes containing uninterrupted ORF in other kinetoplatids: LmjF10.1225 (L. major), LbrM10_V2.1350 (L. braziliensis), Tc00.1047053506153.6 (Trypanosoma cruzi) and Tb927.8.4690 (T. brucei). In spite of this particular finding, as overall conclusion, it must be stated that SIDER2 elements are rare in coding sequences.
On the other hand, several lines of evidence suggest that SIDER2 elements are frequently found in untranslated regions (UTRs) of genes, mainly 3'UTRs. Using both bioinformatics and experimental approaches, Bringaud et al.  demonstrated that SIDER2 elements are present in 3-UTRs of many different genes. Furthermore, these authors showed experimental evidence that SIDER2 sequences are promoting downregulation of mRNA steady state levels. In addition, our database analyses showed that several L. chagasi cDNAs have SIDER2 sequences, reinforcing the idea that these elements are frequently found in UTRs of mRNAs, playing putative regulatory role in gene expression.
Our search on GenBank and EMBL databases showed the existence of SIDER2 elements in other relevant Leishmania genomic regions. For example, homology to SIDER2 sequences is found in a 44-kb genomic region, which was involved in mitotic stability of extrachromoses in L. donovani . To date, the DNA elements participating in the chromosomal replication and segregation processes are largely unknown in Leishmania and other trypanosomatids. The difficulty to uncover the centromeres in trypanosomatids could be pointing to the existence of holocentric chromosomes that are characterized by the presence of a diffuse or nonlocalized centromere during mitosis . In this scenario, SIDER2 elements should be considered as candidates for centromeric sequences. This hypothesis is based on two features of SIDER2 elements: they are distributed regularly along the chromosomes (Figs. 1 and 4) and they have G+C-rich sequences. Richness in G+C-sequences is observed in centromeres and pericentromic regions of many organisms . Also, it is noticeable the existence, within the SIDER2 sequences, of G-rich tracts that are known for their propensity to form G-quadruplex DNA structures .
Finally, the presence of the "79-bp signature" in a large proportion of the SIDER2 elements may be suggestive of a transcriptional role for this class of repeats. In a previous report, we have demonstrated that the "79-bp signature" (also named Pr77), derived from T. cruzi L1Tc non-LTR retrotransposon has a RNA-pol II-dependent promoter that strongly activates gene transcription . In this context, it may be postulated that SIDER2s bearing the "79-bp signature" could be acting as RNA-pol II recruiting points to enhance the transcriptional active at some chromosomal regions.
In this study, we describe several features of a family of novel repeated elements (named SIDER2) that are interspersed along the different chromosomes and present in all Leishmania species. We show an in-depth analysis of these elements in the L. infantum chromosomes 20 and 32, and in the L. major and L. braziliensis chromosomes 32. Apart from their proved role in post-transcriptional regulation of gene expression in Leishmania, our analyses suggest that SIDER2 elements could be involved in DNA amplification phenomena and, perhaps, they can represent centromeric sequences of holocentric chromosomes. In summary, SIDER2 elements constitute a relevant piece of the Leishmania genome organization, and this work provides a framework for investigating the functions of these sequences.
Parasites and DNA isolation
L. infantum JPC strain (MCAN/ES/98/LLM-724, clone M5) was used for arrays construction. For Southern blot analysis, the following Leishmania species were used: L. tropica (MHOM/SU/74/K-27), L. mexicana (MNYC/BZ/62/M-379), L. amazonensis (IFLA/BR/67/PH-8), L. braziliensis (MHOM/BR/75/M-2904) and L. major (MHOM/IL/80/Friedlin). Promastigote forms were cultured in vitro at 26°C in RPMI 1640 medium (Sigma), supplemented with 10% heat-inactivated foetal calf serum (Sigma).
Genomic DNA was prepared from 2 × 108 promastigotes. After washing with phosphate-buffered saline (PBS), cells were suspended in 500 μl of lysis buffer (0.15 M NaCl, 0.1 M EDTA (pH 8.0) and 0.5% SDS). Afterwards, proteinase K was added to a final concentration of 0.1 mg/ml. After incubation for 30 min at 50°C, samples were extracted sequentially with phenol, a phenol-chloroform-isoamyl alcohol (25:24:1) mixture and a chloroform-isoamyl alcohol (24:1) mixture. After adding 0.1 volumes of 3 M sodium acetate and 2.5 volumes of cold ethanol, DNA was collected by centrifugation. The pellet was suspended in 200 μl of Te buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0) and incubated with RNAse A (20 μg/ml final concentration) for 30 min at 37°C. Afterwards, DNA samples were extracted with a phenol-chloroform-isoamyl alcohol (25:24:1) mixture, and DNA precipitated by addition of 0.5 volumes of 7.5 M ammonium acetate and 2 volumes of cold ethanol. Finally, DNA was suspended in 100 μl of Te buffer.
L. infantum genomic arrays
Genomic DNA macroarrays were constructed as previously described . Briefly, a genomic library of Sau 3AI DNA fragments (4-kb average size) was constructed in pBluescript KS plasmid (Promega). DNA from individual colonies was prepared using the Perfectprep Plasmid 96 Vac kit (Eppendorf) and the BIOMEK 2000 robot (Beckam). DNA from 575 different clones was spotted in triplicate onto positively charged nylon membranes (Schleicher and Schuell) by NewBioTechnic (Sevilla, Spain).
Before hybridizations, macroarray membranes were washed with 0.5 M phosphate buffer (pH 7.2) and incubated for 2 h at 65°C in 20 ml of hybridization solution (0.5 M phosphate buffer (pH 7.2), 7% SDS and 1 mM EDTA). For hybridization, 350 ng of L. infantum genomic DNA were labelled by nick-translation using 50 μCi of [α-32P]dCTP (3000 Ci/mmole; Amersham) and standard methods . The labelled-DNA was added to the hybridization solution, and membranes were further incubated for 12 h at 65°C. Afterwards, membranes were washed three times with washing solution (40 mM phosphate buffer (pH 7.2) and 0.1% SDS) for 20 min at 65°C. Radioactive signals were analyzed by a Phosphorimager (Fuji BAS-1500).
DNA sequencing of clone pGLi5-G8g
Both strands of the insert of clone pGLi5-G8g were sequenced using an automated sequencer (ABI Prism 3730; Applied Biosystems) by the Genomics Unit of the Parque Científico de Madrid (SIDI-UAM). Nucleotide sequence of this clone has been deposited at European Molecular Biology Laboratory (EMBL/EBI) nucleotide sequence database under accession number AM937229.
Identification of SIDER2 sequence elements in Leishmania databases
An initial BLASTN search of the L. infantum database  using the sequence of clone pGLi5-G8g showed that this clone contains a genomic region from chromosome 32. However, a subregion of approximately 550-bp was found to be widespread along the L. infantum genome. For the identification of these repeated sequences, now called LiSIDER2 elements, an iterative process was followed. For a given chromosome, sequence blocks showing sequence identity ≥ 60% and length ≥ 100 nucleotides with pGLi5-G8g sequence were considered for further analysis. Selected sequences (plus surrounding upstream and downstream sequences) were aligned using ClustalW. The clustering of sequences into subfamilies was carried out by phylogenetic analysis (see below). To determine the extent of the elements belonging to a given subfamily, the particular sequences were aligned with ClustalW and the extremities determined by visual inspection of the alignment. A subfamily was defined as a group of elements sharing sequence identity ≥ 85%. When the size of an element was clearly different to the medium size for the elements of the subfamily, it was considered as truncated element. Each time a subfamily was identify, the sequence of the longest member of the subfamily was used to perform additional BLASTN searches in the Leishmania databases (contig sequences, ), the retrieved sequences (if new) were aligned as indicated above; the process was repeated until no new sequences were obtained. Finally, the remaining matches, non-assigned to any subfamily, were considered as SIDER2 "orphan" elements. Given the complexity of the identification process, we restricted the analyses to contigs for chromosomes 32 (LinJ32_20070420_V3) and 20 (LinJ20_20070420_V3).
Identification of SIDER2 elements in L. major and L. braziliensis databases  was performed by BLASTN searches using representative members for the LiSIDER2 subfamilies and the orphans LiSIDER2 elements of L. infantum chromosome 32. Each time a homologous sequence was retrieved, it was used to perform additional BLASTN searches in the database. The size and genomic positions for the different LmjSIDER2 or LbSIDER2 elements were determined by sequence alignments using ClustalW and manual corrections. Again, we restricted our analysis to chromosomes 32 of L. major (LmjF32_01_20050601_V5.2) and L. braziliensis (LbrM32, version 2.0).
Multiple alignments and phylogenetic trees
The complete LiSIDER2 sequences were aligned using the default options of ClustalW2 . The resulting alignments were used to perform phylogenetic analysis conducted with the program MEGA version 3.1  using the Neighbour-Joining method and default parameters.
Other databases mining
The different LiSIDER2 elements found in L. infantum chromosome 32 were used for BLASTN searches in T. brucei and T. cruzi databases . Also, BLAST searches were performed in GenBank and EMBL databases.
DNA probes and Southern blot analysis
LiSIDER2-32-121058r and LiSIDER2-20-575257d elements were PCR amplified using as template genomic DNA from L. infantum JPC strain. As primers, the following oligonucleotides were used: LiRS-32-Ad (5'-CCGCCCCGAAATATAAGT-3') and LiRS-32-Ar (5'-GCCTCCATGCGCGGTGTC-3') for LiSIDER2-32-121058r; 20R-d (5'-CCACATCGCGCGTGGCGC-3') and 20R-r (5'-TGACGTGTGGACCCCGCT-3') for LiSIDER2-20-575257d. The amplification products were cloned into the pCR2.1 vector (Invitrogen), yielding clones pLiRS-32A (LiSIDER2-32-121058r) and pLiRS-20Q (LiSIDER2-20-575257d). The authenticity of clones and the fidelity of the PCR-amplification were verified by nucleotide sequencing.
For Southern blot analysis, 1 μg of total DNA from the different Leishmania species was digested with the Sal I restriction enzyme and electrophoresed on 0.8% agarose cells. After ethidium bromide visualization, DNA was transferred to nylon membranes (Hybond-N, Amersham) by standard methods . For probe preparations, Eco RI-inserts of clones pLiRS-32A and pLiRS-20Q were labelled with [α-32P]dCTP by nick-translation . Hybridizations were performed as reported earlier .
Genome sequence data from the Sanger Institute sequencing projects (GeneDB) were invaluable for this work and their provision in the public domain is gratefully acknowledged. Thanks are also given to three anonymous reviewers for their valuable comments. Researchers interested in the sequence datasets described here should contact the authors. This work was funded by grants from the Ministerio de Ciencia y Tecnología (BFU2006-08346), the Instituto de Salud Carlos III (ISCIII-RETIC RD06/0021/0008-FEDER and ISCIII-RETIC RD06/0021/0014-FEDER), and Plan Nacional de I+D+I (BFU2007-65095). Also, an institutional grant from Fundación Ramón Areces is acknowledged.
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