Reverse Transcriptase Domain Research Articles

Characterization of the C-Terminal DNA-binding/DNA Endonuclease Region of a Group II Intron-encoded Protein

Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3′ end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H–N–H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg 2+ ion at the H–N–H active site. Although the conserved cysteine residue pairs could potentially bind Zn 2+, the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn 2+, and the addition of exogenous Zn 2+ inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.

N-terminal domain of yeast telomerase reverse transcriptase: recruitment of Est3p to the telomerase complex.

Telomerase is a reverse transcriptase that maintains chromosome ends. The N-terminal half of the catalytic protein subunit (TERT) contains three functional domains (I, II, and III) that are conserved among TERTs but not found in other reverse transcriptases. Guided by an amino acid sequence alignment of nine TERT proteins, mutations were introduced into yeast TERT (Est2p). In support of the proposed alignment, mutation of virtually all conserved residues resulted in loss-of-function or temperature sensitivity, accompanied by telomere shortening. Overexpression of telomerase component Est3p led to allele-specific suppression of the temperature-sensitive mutations in region I, suggesting that Est3p interacts with this protein domain. As predicted by the genetic results, a lethal mutation in region I resulted in loss of Est3p from the telomerase complex. We conclude that Est2p region I is required for the recruitment of Est3p to yeast telomerase. Given the phylogenetic conservation of region I of TERT, this protein domain may provide the equivalent function in all telomerases.

LINEs and SINE-like elements of the protist Entamoebahistolytica

A survey of whole genome shotgun sequences of the protozoan parasite Entamoeba histolytica revealed three families of non-long terminal repeat (LTR) retrotransposons or long interspersed elements (LINEs) (called EhLINEs in this report). The 4.8 kb EhLINEs each had a single open reading frame with a putative nucleic acid binding motif (CCHC) and restriction enzyme-like endonuclease domain located downstream of the reverse transcriptase (RT) domain. Phylogenetic analysis of the RT domain placed the EhLINEs in the R4 clade of non-LTR elements, a mixed clade of non-LTR elements that includes members from nematodes, insects, and vertebrates. EhLINE1 (which was previously identified as HMc and EhRLE) shared a common 3′ end with a highly transcribed 0.55 kb short interspersed element (SINE)-like element previously identified as IE or ehapt2 and called EhLSINE1 in this report. Similarly, EhLINE2 shared a common 3′ end with a highly transcribed 0.65 kb SINE-like element called EhLSINE2 in this report. The shared 3′ end sequences of the EhLINEs and EhLSINEs suggested that EhLINEs are involved in the retrotransposition of the EhLSINEs. EhLSINEs were flanked by target site duplications and contained conserved 5′ sequences, which likely regulate their transcription. The EhLSINEs are the first protist SINE-like elements identified that share a common 3′ sequence with LINEs, and the first SINE-like elements that have been associated with the R4 clade of non-LTR elements.

Functional Analysis of the C-terminal Extension of Telomerase Reverse Transcriptase: A PUTATIVE “THUMB” DOMAIN

Telomerase is an RNA-protein complex responsible for the extension of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to reverse transcriptases (RTs) encoded by retroviruses and retroelements. The mechanisms of telomerase may therefore be similar to those of the conventional reverse transcriptases. In this report, we explore potential similarity between these two classes of proteins in a region with no evident sequence similarity. Previous analysis has implicated a C-terminal domain of retroviral RTs (known as the "thumb" domain) in template-primer binding and in processivity control. The equivalent region of TERTs, although similar to one another, does not exhibit significant sequence homology to retroviral RTs. However, we found that removal of this region of yeast TERT similarly resulted in a decrease in the stability of telomerase-DNA complex and in the processivity of telomerase-mediated nucleotide addition. Moreover, the C-terminal domain of TERT exhibits a nucleic acid binding activity when recombinantly expressed and purified. Finally, amino acid substitutions of conserved residues in this region of TERT were found to impair telomerase activity and processivity. We suggest that mechanistic similarity between telomerase and retroviral RTs may extend beyond the regions with apparent sequence similarity.

Evaluation of telomerase mRNA (hTERT) in colon cancer.

Telomerase activation, a cardinal requirement for immortalization, is a crucial step in the development of malignancy and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. By reverse transcription-PCR, using primers within the reverse transcriptase domain of hTERT, we investigated telomerase messenger in 8 adenomatous and 9 dysplastic polyps, and in 32 paired cancer-normal mucosa specimens, one liver and one spleen metastasis from patients resected for sporadic colorectal cancer. Telomerase messenger was absent or very low in normal mucosa and in adenomatous polyps. Dysplastic polyps and adenocarcinoma samples showed hTERT mRNA, with higher levels in cancer tissues compared to dysplastic lesions. A high telomerase messenger level was shown to be associated with late-staged cancers and with metastasis; thus, detection of telomerase messenger may be useful in the early diagnosis of colon cancer, and telomerase may be a new target for therapeutic intervention.

Bali1A, a Possible LINE Partner for the SINE S1 in <I>Brassica napus</I>

SINEs (short interspersed elements) are nonautonomous retroposons that may use the enzymatic machinery of autonomous LINEs (long interspersed elements) for retroposition. We have iso- lated a potentially active LINE element in Brassica napus. This 5.4-kb LINE, named Bali1A, is the only full-length member of a small LINE family composed of three copies per haploid genome. Bali1A ends with a poly(A) region, is flanked by direct repeats, and possesses two uninterrupted open reading frames (ORFs) characteristic of LINEs: an ORF1 presenting the cysteine-rich motifs and an ORF2 showing strong similarities to reverse transcriptase and endonuclease. A phylogenetic comparison between the endonu- clease and reverse transcriptase domain of Bali1A and other LINEs allowed its classification in the L1 clade. Bali1A transcripts were detected in Brassica napus, but its 5 � -UTR is not able to drive the expres- sion of a reporter gene in Arabidopsis thaliana. Although no primary sequence homology exists between Bali1A and S1, the perfect association in the Cruciferae family (46 species tested) of the LINE Bali1 family with the SINE S1 family suggests that Bali1A may be the retroposition partner of S1. In that case, the Bali1A/S1 association could depend on a shared poly(A) 3 � region as proposed for the L1/Alu association in humans.

Sequence variation and genomic amplification of a family of Gypsy-like elements in the oomycete genus Phytophthora.

A family of sequences resembling Gypsy retroelements was identified and shown to be widely distributed throughout the genus Phytophthora, a member of the algallike oomycete fungi. Polymerase chain reaction (PCR) using specific and degenerate primers detected the family in 29 of 37 species tested. DNA hybridization also failed to detect the sequences in the eight species that were negative in PCR. The element appears to have been a major force in the shaping of Phytophthora genomes because its abundance varied drastically from about 10 to more than 10,000 copies per genome within the species containing the element. Family members diverged from each other by single-base changes, insertions, and deletions, with a mean nucleotide divergence of 16.7%. By constructing phylogenies of the elements, lineages were identified that predated speciation events within Phytophthora and subfamilies that had diverged more recently. The element was studied in detail in Phytophthora infestans, in which about 30 copies are dispersed throughout the genome. Phylogenetic comparisons of the reverse transcriptases placed the family within the Ty3/Gypsy group of long terminal repeat (LTR) retrotransposons, with the closest affinities to elements from plants. However, each of 12 family members sequenced contained defects that would render their protein products inactive, including frameshift mutations within reverse transcriptase domains and truncations that appeared to eliminate gag, protease, and terminal repeat sequences.

Mutation of the catalytic domain of the foamy virus reverse transcriptase leads to loss of processivity and infectivity.

Foamy virus (FV) replication is resistant to most nucleoside analog reverse transcriptase (RT) inhibitors. In an attempt to create a 2',3'-dideoxy-3'-thiacytidine (3TC)-sensitive virus, the second residue in the highly conserved YXDD motif of simian foamy virus-chimpanzee (human isolate) [SFVcpz(hu)] RT was changed from Val (V) to Met (M). Unexpectedly, the resultant virus, SFVcpz(hu) RT-V313M, replicated poorly, and Met rapidly reverted to Val. Despite the presence of approximately 50% of wild-type RT activity in RT-V313M virions, full-length DNA products were not detected in transfected cells. Using purified recombinant enzymes, we found that the wild-type FV RT is significantly more processive than human immunodeficiency virus type 1 RT. However, the V313M mutant has about 40% of the wild-type level of FV RT activity and has a lower processivity than the wild-type FV enzyme. The V313M mutant RT is also relatively resistant to 3TC. These results suggest that the decrease in RT activity and processivity of FV RT-V313M prevents completion of reverse transcription and greatly diminishes viral replication.

Resistance surveillance in chronic hepatitis B patients treated with adefovir dipivoxil for up to 60 weeks

Current therapies for chronic hepatitis B virus (HBV) infection do not provide adequate long-term control of viral replication in the majority of patients. Monotherapy with nucleoside analogs, such as lamivudine and famciclovir, is effective for short periods but results in the emergence of drug-resistant HBV in a substantial number of patients within 1 year of therapy. Adefovir dipivoxil (ADV) has demonstrated clinical activity against wild-type and lamivudine-resistant HBV, but it is unclear whether resistance mutations will emerge after long-term therapy with this drug. To determine whether extended treatment with ADV led to the emergence of drug-resistant populations of HBV, we analyzed virus isolated from patients currently enrolled in a long-term open-label study. The reverse transcriptase domain of HBV polymerase was amplified and sequenced from patients that had received a cumulative exposure of up to 60 weeks of ADV. During our analyses, several previously unreported amino acid substitutions were observed in the reverse transcriptase domain of HBV. Importantly, none of the observed mutations occurred in more than 1 patient, nor were they associated with an adefovir-resistant phenotype in vitro. Furthermore, none of the patients from whom these mutant viruses were isolated had evidence of virologic rebound. In conclusion, these results, although based on a limited number of patients, suggest that treatment with ADV does not lead to the emergence of resistant virus after up to 60 weeks of therapy. (H EPATOLOGY 2002;36:464-473.)

Development of a monoclonal antibody for neutralization of human telomerase activity.

To develop monoclonal antibodies (MAbs) to neutralize human telomerase, an epitope (hTERT(7)) in reverse transcriptase domain of hTERT was synthesized and was used to immunize BALB/c mice. Hybridomas were generated and screened by enzyme-linked immunoadsorbent assay (ELISA) for specific MAbs. One hybridoma M2 clone, isotyped IgG(1), was established. The competitive assay confirmed that the M2 antibody was hTERT(7) specific, and the affinity constant was about 1 x 10(6) M(-1). M2 could recognize cell extracts from HeLa cancer cells but not those of normal 2BS cells in ELISA assay. For in situ staining immunohistochemically, the positive staining presented in the nuclear compartment of HeLa, while 2BS was nonreactive. In TRAP-PCR ELISA, M2 markedly decreased the activities of human telomerase in HeLa cells. The sequencing of M2 heavy chain variable region proved its mouse origin. The results demonstrated that the developed mouse MAb should be hTERT specific and could not only recognize native cellular hTERT in ELISA and immunohistochemistry, but also neutralize telomerase activities. Thus, it could be hoped that the antibody could be used in clinical diagnosis and treatment of cancers.

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo.

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.

Mutating Conserved Residues in the Ribonuclease H Domain of Ty3 Reverse Transcriptase Affects Specialized Cleavage Events

The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts. However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1. In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT. The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae. Although all mutations had minimal impact on DNA polymerase function, amidation of Asp-358, Glu-401, and Asp-426 eliminated Mg(2+)- and Mn(2+)-dependent RNase H function. Replacing His-427 and Tyr-459 with Ala and Asp-469 with Asn resulted in reduced RNase H activity in the presence of Mg(2+), whereas in the presence of Mn(2+) these mutants displayed a lack of turnover. Despite this, mutations at all positions were lethal for transposition. To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme. Mutants retaining RNase H activity on a heteropolymeric RNA.DNA hybrid failed to support DNA strand transfer and release of the (+) strand polypurine tract primer from (+) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.

Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the specific interaction between the RT and a viral RNA signal termed epsilon, which bears the specific template sequence for protein priming. The product of protein priming is a short oligodeoxynucleotide which represents the 5' end of the viral minus-strand DNA and is covalently attached to the RT. We have now identified truncated RT variants from the duck hepatitis B virus that were fully active in the initial step of protein priming, i.e., the covalent attachment of the first nucleotide to the protein (RT deoxynucleotidylation), but defective in any subsequent DNA polymerization. A short sequence in the RT domain was localized that was dispensable for RT deoxynucleotidylation but essential for the subsequent DNA polymerization. These results have thus revealed two distinct stages of protein priming, i.e., the initial attachment of the first nucleotide to the RT (RT deoxynucleotidylation or initiation of protein priming) and the subsequent DNA synthesis (polymerization) to complete protein priming, with the second step entailing additional RT sequences. Two models are proposed to explain the observed differential sequence requirement for the two distinct stages of the protein priming reaction.

Hepatitis B virus resistance to lamivudine and its clinical implications.

Lamivudine is the first orally available drug approved for treatment of chronic hepatitis B, but hepatitis B virus (HBV) resistance to lamivudine appears to be a sine qua non in the therapy of HBV. The mutations at the FLLA and YMDD motif in the domains B and C of HBV polymerase contribute to this resistance. These mutations are found at codon (or AA) rtL180M and rtM204V/I in the reverse transcriptase (RT) domain of the HBV polymerase for all genotypes according to a new standardized RT domain numbering system. The resistant HBV may be less replication-competent in vitro and in vivo, and it is rarely associated with markedly increased HBV replication or liver injury. Therefore, certain physicians favour continuing lamivudine therapy even after emergence of HBV resistance with the expectation of maintaining lower-than baseline HBV DNA, alanine amino-transferase, and histological improvement, and avoiding reversion to wild-type HBV until additional antiviral strategies are developed. Ultimately, once several antiviral agents are approved, combination strategy is likely to be incorporated in antiviral treatment for chronic HBV to suppress, prevent or minimize the emergence of resistant virus.

Ylli, a Non–LTR Retrotransposon L1 Family in the Dimorphic Yeast Yarrowia lipolytica

During the course of a random sequencing project of the genome of the dimorphic yeast Yarrowia lipolytica, we have identified sequences that were repeated in the genome and that matched the reverse transcriptase (RT) sequence of non-long terminal repeat (non-LTR) retrotransposons. Extension of sequencing on each side of this zone of homology allowed the definition of an element over 6 kb long. The conceptual translation of this sequence revealed two open reading frames (ORFs) that displayed several characteristics of non-LTR retrotransposons: a Cys-rich motif in the ORF1, an N-terminal endonuclease, a central RT, and a C-terminal zinc finger domain in the ORF2. We called this element Ylli (for Y. lipolytica LINE). A total of 19 distinct repeats carrying the 3' untranslated region (UTR) and all ending with a poly-A tail were detected. Most of them were very short, 17 being 134 bp long or less. The number of copies of Ylli was estimated to be around 100 if these short repeats are 5' truncations. No 5' UTR was clearly identified, indicating that entire and therefore active elements might be very rare in the Y. lipolytica strain tested. Ylli does not seem to have any insertion specificity. Phylogenetic analysis of the RT domain unambiguously placed Ylli within the L1 clade. It forms a monophyletic group with the Zorro non-LTR retrotransposons discovered in another dimorphic yeast Candida albicans. BLAST comparisons showed that ORF2 of Ylli is closely related to that of the slime mold Dictyostelium discoideum L1 family, TRE.

Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosomajaponicum

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is ∼3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5′- and 3′-terminal untranslated regions and, at its 3′-terminus, SjR2 bears a short (TGAC) 3 repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 from the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that ∼10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5′-untranslated region of SjR2 was >80% identical to the 3′-untranslated region of a schistosome heat shock protein-70 gene ( hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni.

Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in Gag-Pol are not essential for incorporation of the Pr160(gag-pol) into virus particles.

Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55(gag). Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminal gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.

A novel genetic algorithm for designing mimetic peptides that interfere with the function of a target molecule.

We designed a new computer program (MIMETIC), which generates a series of peptides for interaction with a target peptide sequence. The genetic algorithm employed ranks the sequences obtained from one generation to the next by "goodness of fit" to the target. MIMETIC designed recognition peptides to various regions of HIV-1 reverse transcriptase. Among ten peptide candidates synthesized, three inhibited reverse transcription in vitro. TLMA2993 and PSTW1594 both targeted the connection domain of reverse transcriptase and ESLA2340 targeted the thumb domain.

Xena, a full-length basal retroelement from tetraodontid fish.

Mobile genetic elements are ubiquitous throughout the eukaryote superkingdom. We have sequenced a highly unusual full-length retroelement from the Fugu fish, Takifugu rubripes. This element, which we have named Xena, is similar in structure and sequence to the Penelope retroelement from Drosophila virilis and consists of a single long open reading frame containing a reverse transcriptase domain flanked by identical direct long terminal repeat (LTR) sequences. These LTRs show an organization similar to the terminal repeats already described in the Penelope retrotransposon of Drosophila but are structurally and functionally distinct from the LTRs carried by LTR-retrotransposons. In view of their distinctness, we refer to these repeats as PLTRs (Penelope-LTRs). Whereas the element contains a reverse transcriptase, no other domains or motifs commonly associated with retroelements are present. In the full-length Fugu element, the 5' direct PLTR is preceded by an inverted PLTR fragment. Additional elements, many showing various degrees of deletion, are described from the Fugu genome and from that of the freshwater pufferfish Tetraodon nigroviridis. Many of these additional elements are also preceded by inverted PLTR sequences. Xena-like elements are also described from the genomes of several other organisms. The Penelope-Xena lineage is apparently a basal group within the retrotransposons and therefore represents an evolutionarily important class of retroelement.

Genomic and chromosomal organization of Ty1- copia-like sequences in Olea europaea and evolutionary relationships of Olea retroelements.

The Ty1- copia-like retrotransposon is one of the commonest class of transposable elements in the plant kingdom, often comprising several percent of the total DNA content. We aimed to study the evolutionary relationships of Olea retroelements, using part of the reverse transcriptase domain, as well as the genomic and chromosomal organization of these sequences in Olea europaea chromosomes and their transcription activity and copy number. Fourteen clones, that were isolated from four different species, were sequenced and a phylogenetic tree was constructed based on their predicted amino acids. Five clones derived from O. europaea were clustered together with a 87% nucleotide sequence homology and two Olea oleaster clones showed 98% sequence homology. The rest of the clones showed heterogeneity among them, leading to a common ancestral transposon that existed before the genus arose. The Ty1- copia-like sequences have a dispersed genomic organization, physically distributed on all chromosomes, showing minor clustering in some cases and low copy numbers in the smallest chromosome pair. The total copy number in the O. europaea genome was estimated by dot blotting to be 40,000 in a haploid nucleus, but a number of these are non-functional since the sequenced clones contained stop codons and frame-shifts. Some Ty1- copia-like copies, present in O. europaea, were found to be methylated, while no differences in methylation were observed between DNA isolated from young leaves and callus-suspension cultures.