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ABSTRACT: The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state.
Fungal Genetics and Biology 03/2012; 49(3):250-61. · 3.74 Impact Factor
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ABSTRACT: The process of decomposition of bodies in the marine environment is poorly understood and almost nothing is currently known about the microorganisms involved. This study aimed to investigate the microbes involved in decomposition in the sea and to evaluate the potential use of marine bacterial succession for postmortem submersion interval (PMSI) estimation, for which there is currently no reliable method. Partial pig remains were completely submerged during autumn and winter and were regularly sampled to document marine bacterial colonisation and the changes in community composition over time. Five stages of decomposition were recognised, some of which exhibited characters specific for partial carrion. Marine bacteria rapidly colonised the submerged remains in a successional manner. Seasonal differences were observed for the rate of decomposition and also for several groups of colonising bacteria. Marine bacteria specific for particular PMSIs were identified. This study provides an insight into the involvement of saprophytic marine bacteria in the decomposition of mammalian remains in the sea and is the first to explore the use of marine bacterial colonisation and succession as a novel tool for PMSI estimation. We propose that with further study, marine bacterial succession will prove useful for determination of the length of time a body may have been immersed in a marine environment.
Forensic science international 11/2010; 209(1-3):1-10. · 2.10 Impact Factor
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ABSTRACT: Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/-). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/- heterozygote, the homing endonuclease initiates intein 'homing' by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.
Fungal Genetics and Biology 04/2010; 47(4):392-8. · 3.74 Impact Factor
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ABSTRACT: As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the alpha-complementation peptide abolished the peptide's ability to restore beta-galactosidase activity, while an active variant allowed complementation. This alpha-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a beta-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.
Biochimica et Biophysica Acta 09/2007; 1774(8):995-1001. · 4.66 Impact Factor
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ABSTRACT: The dependence of protein splicing on conserved residues of the Cne PRP8 intein was assessed by alanine scanning mutagenesis in a foreign protein context. Corroboration was obtained for the involvement of residues at the splice junctions and of the conserved threonine and histidine of motif B. Five additional residues were identified as absolutely required for splicing. Variant W151A displayed premature C-terminal cleavage, not seen with other Cne PRP8 mutants. We propose a model whereby W151 acts to prevent premature C-terminal cleavage, favoring complete splicing as opposed to two disjointed cleavage events.
FEBS Letters 07/2007; 581(16):3000-4. · 3.54 Impact Factor
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ABSTRACT: An intein is a protein sequence embedded within a precursor protein that is excised during protein maturation. Inteins were first found encoded in the VMA gene of Saccharomyces cerevisiae. Subsequently, they have been found in diverse organisms (eukaryotes, archaea, eubacteria and viruses). The VMA intein has been found in various saccharomycete yeasts but not in other fungi. Different inteins have now been found widely in the fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins. A protein distantly related to inteins, but closely related to metazoan hedgehog proteins, has been described from Glomeromycota. Many of the newly described inteins contain homing endonucleases and some of these are apparently active. The enlarged fungal intein data set permits insight into the evolution of inteins, including the role of horizontal transfer in their persistence. The diverse fungal inteins provide a resource for biotechnology using their protein splicing or homing endonuclease capabilities.
Fungal Genetics and Biology 04/2007; 44(3):153-79. · 3.74 Impact Factor
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ABSTRACT: Target-primed (non-LTR) retrotransposons, such as the human L1 element, are mobile genetic elements found in many eukaryotic genomes. They are often present in large numbers and their retrotransposition can cause mutations and genomic rearrangements. Despite their importance, many aspects of their replication are not well understood.
We have developed a yeast model system for studying target-primed retrotransposons. This system uses the Zorro3 element from Candida albicans. A cloned copy of Zorro3, tagged with a retrotransposition indicator gene, retrotransposes at a high frequency when introduced into an appropriate C. albicans host strain. Retrotransposed copies of the tagged element exhibit similar features to the native copies, indicating that the natural retrotransposition pathway is being used. Retrotransposition is dependent on the products of the tagged element's own genes and is highly temperature-regulated. The new assay permits the analysis of the effects of specific mutations introduced into the cloned element.
This Zorro3 retrotransposition assay system complements previously available target-primed retrotransposition assays. Due to the relative simplicity of the growth, manipulation and analysis of yeast cells, the system should advance our understanding of target-primed retrotransposition.
BMC Genomics 02/2007; 8:263. · 4.07 Impact Factor
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ABSTRACT: Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.
We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2) from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2) of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2), one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins.
The identification of these new inteins increases the known host range of intein sequences in eukaryotes, and provides fresh insights into their origins and evolution. We conclude that inteins are ancient eukaryote elements once found widely among microbial eukaryotes. They persist as rarities in the genomes of a sporadic array of microorganisms, occupying highly conserved sites in diverse proteins.
BMC Biology 02/2006; 4:38. · 5.75 Impact Factor
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ABSTRACT: We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.
In total, 22 PRP8 inteins have been detected in species from three different orders of euascomycetes, including Aspergillus nidulans and Aspergillus fumigatus (Eurotiales), Paracoccidiodes brasiliensis, Uncinocarpus reesii and Histoplasma capsulatum (Onygales) and Botrytis cinerea (Helotiales). These inteins are all at the same site in the PRP8 sequence as the original Cryptococcus neoformans intein. Some of the PRP8 inteins contain apparently intact homing endonuclease domains and are thus potentially mobile, while some lack the region corresponding to the homing endonuclease and are thus mini-inteins. In contrast, no mini-inteins have been reported in the VMA gene of yeast. There are several examples of pairs of closely related species where one species carries the PRP8 intein while the intein is absent from the other species. Bio-informatic and phylogenetic analyses suggest that many of the ascomycete PRP8 homing endonucleases are active. This contrasts with the VMA homing endonucleases, most of which are inactive.
PRP8 inteins are widespread in the euascomycetes (Pezizomycota) and apparently their homing endonucleases are active. There is no evidence for horizontal transfer within the euascomycetes. This suggests that the intein is of ancient origin and has been vertically transmitted amongst the euascomycetes. It is possible that horizontal transfer has occurred between the euascomycetes and members of the basidiomycete genus Cryptococcus.
BMC Evolutionary Biology 02/2006; 6:42. · 3.52 Impact Factor
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ABSTRACT: Only two nuclear encoded inteins have been described. The first, SceVMA, was found in a vacuolar ATPase gene of Saccharomyces cerevisiae and related yeasts. The second, CnePRP8, was found in the PRP8 gene of Cryptococcus neoformans. CnePRP8 contains protein sequences associated with intein splicing but no endonuclease domain. We compared allelic mini-inteins in both varieties of C. neoformans (var. neoformans and var. grubii) and in the related primary pathogen C. gattii to study the evolution of both the mini-intein and the host. We also describe a full-length, endonuclease-containing intein in Cryptococcus laurentii, a moderately distant relation of C. neoformans. We did not detect an intein in the PRP8 gene of other species of Cryptococcus including species closely related to the C. neoformans/C. gattii group. It is therefore probable that the C. neoformans/C. gattii mini-intein was derived from horizontal transfer in which C. laurentii or another intein-containing species was the source.
Fungal Genetics and Biology 06/2005; 42(5):452-63. · 3.74 Impact Factor
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ABSTRACT: Until recently the only intein known to be encoded by the nuclear genome of a eukaryote was the VMA intein in the vacuolar ATPase precursor of several species of saccharomycete yeast. This intein has been intensively studied and much information has been gained about its structure, mode of action and evolutionary history. We recently reported a second nuclear intein, Cne PRP8, encoded within the PRP8 gene of the basidiomycete Cryptococcus neoformans. Subsequent studies have found allelic PRP8 inteins in several species of yeast and filamentous ascomycetes. Here we report two further, non-allelic, inteins from ascomycete species. The yeast Debaryomyces hansenii (which also has a VMA intein) has an intein encoded within the sequence of the glutamate synthase gene (GLT1). There are also inteins encoded in the homologous GLT1 genes of the yeast Candida (Pichia) guilliermondii and the filamentous fungus Podospora anserina. These allelic GLT1 inteins occupy exactly the same site in the glutamate synthase and all contain domains that indicate the presence of a homing endonuclease (HEG). Podospora anserina, in addition, contains a second, non-allelic, intein encoded in the chitin synthase gene (CHS2); this intein also contains a HEG domain. We describe the phylogenetic relationships among the four eukaryote nuclear encoded inteins (VMA, PRP8, GLT1 and CHS2). We also consider this phylogeny in the broader context of eubacterial, archaeal and eukaryote viral and organelle inteins.
Yeast 05/2005; 22(6):493-501. · 1.89 Impact Factor
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ABSTRACT: A wide variety of novel tyrosine recombinase (YR)-encoding retrotransposons were identified using data emerging from the various eukaryotic genome sequencing projects. Although many of these elements are clearly members of the previously described DIRS group of YR retrotransposons, a substantial number, including elements from a variety of fungi and animals, belong to a distinct and previously unrecognized group. We refer to these latter elements as the Ngaro group after a representative from zebrafish. Like the members of the DIRS group, Ngaro elements encode proteins bearing reverse transcriptase (RT) and ribonuclease H (RH) domains similar to those of long terminal repeat (LTR) retrotransposons. Phylogenetic analyses based on alignments of RT/RH and YR domains, however, indicate that Ngaro and DIRS are anciently diverged groups. Differences in coding capacity also support the distinction between the two groups. For instance, we found that DIRS elements all encode a protein domain which is similar in sequence to the DNA methyltransferases of certain bacteriophages, whereas this domain is absent from all Ngaro elements. Together, the Ngaro and DIRS groups of YR retrotransposons contain elements with an astonishing diversity in structures, with variations in the nature of the associated repeat sequences and in the arrangement and complement of coding regions. In addition they contain elements with some surprising features, such as spliceosomal introns and long overlapping open reading frames.
Molecular Biology and Evolution 05/2004; 21(4):746-59. · 5.55 Impact Factor
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ABSTRACT: A new group of transposable elements, which the authors have named cryptons, was detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. These elements are unlike any previously described transposons. An archetypal member of the group, crypton Cn1, is 4 kb in length and is present at a low but variable copy number in a variety of C. neoformans strains. It displays interstrain variations in its insertion sites, suggesting recent mobility. The internal region contains a long gene, interrupted by several introns. The product of this gene contains a putative tyrosine recombinase near its middle, and a region similar in sequence to the DNA-binding domains of several fungal transcription factors near its C-terminus. The element contains no long repeat sequences, but is bordered by short direct repeats which may have been produced by its insertion into the host genome by recombination. Many of the structural features of crypton Cn1 are conserved in the other known cryptons, suggesting that these elements represent the functional forms. The presence of cryptons in ascomycetes and basidiomycetes suggests that this is an ancient group of elements (>400 million years old). Sequence comparisons suggest that cryptons may be related to the DIRS1 and Ngaro1 groups of tyrosine-recombinase-encoding retrotransposons.
Microbiology 11/2003; 149(Pt 11):3099-109. · 3.06 Impact Factor
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ABSTRACT: Helitrons, a novel class of eukaryote mobile genetic elements, are distinguished from other transposable elements by encoding a 'rolling circle' replication (RCR) protein (Rep) and a helicase. Helitrons have recently been described from Arabidopsis, rice and the nematode Caenorhabditis. We now report the discovery of Helitron-like elements in vertebrates, specifically in the genomes of the fish Danio rerio and Sphoeroides nephelus. We also describe Helitrons from the white rot fungus Phanerochaete chrysosporium and from the Anopheles genome. Many of the fish Helitrons have an uncorrupted open reading frame encoding both the RCR Rep protein and a helicase. These fish elements are of particular interest because they also encode, within the single open reading frame, an apurinic-apyrimidinic (AP) endonuclease most closely related to those of certain non-long terminal repeat retrotransposons. As they invariably carry an endonuclease and also form a very distinct clade, we have named these vertebrate elements 'helentrons'. It is likely that these helentrons are still active.
Gene 09/2003; 313:201-12. · 2.34 Impact Factor
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ABSTRACT: Ty3/gypsy retrotransposons are a widespread group of eukaryote mobile genetic elements. They are similar in structure to, and may be ancestors of, the vertebrate retroviruses. Here we describe the first Ty3/gypsy retrotransposons from the pathogenic yeasts Candida albicans and Candida dubliniensis, which we refer to as Tca3 and Tcd3, respectively. Tca3 was first identified in a variety of strains as an element lacking a large part of its coding region. Comparative analyses between C. albicans and C. dubliniensis allowed us to identify the closely related full-length Tcd3 element, and, subsequently, the full-length Tca3 elements. The full-length versions of Tca3 and Tcd3 are broadly similar in structure to other Ty3/gypsy elements, but have several features of special interest, e.g. both elements appear to have a novel mechanism for priming minus-strand DNA synthesis, probably involving conserved secondary structures adjacent to the 5' LTRs. Also, while closely related to each other, the two elements appear to be fairly distantly related to other known Ty3/gypsy-like elements. Finally, the occurrence of the internally deleted forms of Tca3 in many strains raises interesting questions concerning the evolution of these transposable elements in Candida and the evolution of Candida itself. The sequences reported in this paper have been assigned GenBank Accession Nos AF499463, AF499464 and AF510498.
Yeast 05/2003; 20(6):493-508. · 1.89 Impact Factor
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ABSTRACT: 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.
Molecular Biology and Evolution 04/2002; 19(3):247-55. · 5.55 Impact Factor
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ABSTRACT: We have used comparative sequence analysis to identify an intein-like sequence (protein splicing element) present in Cryptococcus neoformans, a fungal pathogen of humans. The sequence encoding this element is present in the C. neoformans PRP8 gene, as an in-frame insertion relative to the PRP8 genes of other organisms. It contains sequences similar to those of the protein-splicing domains of two previously described yeast inteins (in Saccharomyces cerevisiae and Candida tropicalis), although it lacks any recognizable internal endonuclease domain. The Cryptococcus neoformans intein (Cne PRP8) is only the second to be found in a eukaryote nuclear genome; the previously described yeast inteins occur at the same site in the VMA gene homologues of S. cerevisiae and C. tropicalis. The host gene of the Cryptococcus intein, PRP8, encodes a highly conserved mRNA splicing protein found as part of the spliceosome. The Cne PRP8 intein may be a useful drug target in addressing the cryptococcal infections so prevalent in AIDS patients. The Cne PRP8 coding sequence and the flanking sequences of the Cryptococcus neoformans PRP8 gene have been assigned GenBank Accession No. AF349436. Copyright © 2001 John Wiley & Sons, Ltd.
Yeast 10/2001; 18(15):1365 - 1370. · 1.89 Impact Factor
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ABSTRACT: We have undertaken an analysis of the retrotransposons in the medically important basidiomycetous fungus Cryptococcus neoformans. Using the data generated by a C. neoformans genome sequencing project at the Stanford Genome Technology Center, 15 distinct families of LTR retrotransposons and several families of non-LTR retrotransposons were identified. Members of at least seven families have transposed recently and are probably still active. For several families, only partial elements could be identified and these are quite diverse in sequence, suggesting that they are ancient components of the C. neoformans genome. Most C. neoformans elements are not closely related to previously identified fungal retrotransposons, suggesting that the diversity of fungal retrotransposons has been only sparsely sampled to date. C. neoformans has fewer distinct retrotransposon families than Candida albicans (37 or more), in particular fewer families represented solely by ancient and inactive elements, but it has considerably more families than either Saccharomyces cerevisiae (five) or Schizosaccharomyces pombe (two). The findings suggest that elimination of retrotransposons is faster in C. neoformans than in C. albicans, but perhaps not as rapid as in S. cerevisiae or Sz. pombe. The identification of the retrotransposons of C. neoformans should assist in the molecular characterization of this important pathogen, and also further our understanding of the role played by retroelements in genome evolution. Copyright © 2001 John Wiley & Sons, Ltd.
Yeast 06/2001; 18(9):865 - 880. · 1.89 Impact Factor
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ABSTRACT: Non-LTR retrotransposons (also known as LINEs) have had a major influence on the structure of many eukaryote genomes. They
are abundant in many multicellular eukaryotes, including mammals, but appear to be absent from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This absence has, to date, precluded the development of a yeast model system for the study of non-LTR retrotransposons.
In this report we describe several families of non-LTR retrotransposons from the yeast Candida albicans. These elements, which we call Zorro elements, are all members of the L1 clade of non-LTR retrotransposons. Some are intact,
transcriptionally active and have transposed recently. This finding should allow the development of a yeast model system.
Current Genetics 03/2001; 39(2):83-91. · 2.56 Impact Factor
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ABSTRACT: This report describes the identification and characterization of a retrotransposon, termed Tca5, from the pathogenic yeast Candida albicans. Tca5 has identical 685 bp LTRs flanking 4218 bp of internal sequence within which lies a single long ORF. Immediately internal to the left LTR is a primer binding site complementary to an internal portion of the initiator methionine tRNA and upstream of the right LTR is a polypurine tract. The ORF predicts a protein containing all the conserved motifs characteristic of Gag, protease, integrase, reverse transcriptase and RNaseH. Genomic Southern blots probed with Tca5 sequences show that it is a low copy number element and is present at different loci in different strains. This, together with the apparently intact structure of Tca5, suggests that it has transposed very recently. Potentially full-length Tca5 transcripts were detected in some strains raising the possibility that some copies of Tca5 may still be active. Phylogenetic analyses and other sequence comparisons suggest that Tca5 is most closely related to the Ty5 element of Saccharomyces cerevisiae and S. paradoxus. The nucleotide sequence of Tca5 has been submitted to GenBank under Accession No. AF093417. Copyright © 2000 John Wiley & Sons, Ltd.
Yeast 11/2000; 16(16):1509 - 1518. · 1.89 Impact Factor
