DIRS-1 and the other tyrosine recombinase retrotransposons.

DIRS-1 is a retroelement from the slime mold Dictyostelium discoideum. Until recently only two related retrotransposons had been described: PAT from the nematode Panagrellus redivivus and Prt1 from the zygomycete fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these three elements suggested that they were closely related to each other and more distantly related to the Ty3/gypsy Long Terminal Repeat (LTR) retroelements. They have several unusual structural features that distinguish them from typical LTR elements. For instance, they each encode a tyrosine recombinase (YR), but not a DDE-type integrase or an aspartic protease. Although the DIRS-1-related elements are bordered by terminal repeats these differ from typical LTRs in a number of ways. In DIRS-1, for example, the terminal repeats are inverted (complementary), non-identical in sequence, and the outer edges of the terminal sequences are repeated (adjacent to each other) in the internal region. PAT has so-called "split" direct repeats in which the unrelated terminal sequences appear as direct repeats adjacent to each other in the internal region. The only repetition displayed by Prt1 is the presence of short inverted terminal repeats, but the sequenced copy of this element is believed to be a truncated version of an element with a structure resembling DIRS-1. The unusual structure of the terminal repeats of the DIRS1-like elements appears to be related to their replication via free circular intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. In recognition of these important distinctions it is proposed that the retrotransposons that encode tyrosine recombinases be called the tyrosine recombinase (or YR) retrotransposons. Recently a large number of additional YR retrotransposons have been described, including elements from fungi (zygomycetes and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish and amphibia, while remnants of elements related to DIRS-1 occur in the human genome. The complete set of YR retrotransposons can be divided into two major groups, the DIRS elements and the Ngaro elements, the two groups forming distinct clades on phylogenetic trees based on alignments of RT/RH and recombinase sequences, and also having some structural distinctions. A third group of transposable elements, which we call Cryptons, also carry tyrosine recombinases. These elements do not encode a reverse transcriptase and so are believed to be DNA transposons not retrotransposons. They have been detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. Sequence comparisons suggest that the Crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a Crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon.

DIRS retroelements in arthropods: identification of the recently active TcDirs1 element in the red flour beetle Tribolium castaneum.

Members of the DIRS family of retrotransposons differ from most other known retrotransposons in that they encode a tyrosine recombinase (YR), a type of enzyme frequently involved in site-specific recombination. This enzyme is believed to insert the extrachromosomal DNA intermediate of DIRS element retrotransposition into the host genome. DIRS elements have been found in plants, a slime mold, fungi, and a variety of animals including vertebrates, echinoderms and nematodes. They have a somewhat patchy distribution, however, apparently being absent from a number of model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster. In this report we describe the first DIRS retroelement to be identified in an arthropod. This element, TcDirs1, was found in the red flour beetle Tribolium castaneum (Coleoptera). It is generally similar in sequence and structure to several previously described members of the DIRS group: it is bordered by inverted terminal repeats and it has a similar set of protein-coding domains (Gag, reverse transcriptase/ribonuclease H, and the YR), although these are arranged in a novel fashion. TcDirs1 elements exhibit several features indicative of recent activity, such as intact coding regions, a high level of sequence similarity between distinct elements and polymorphic insertion sites. Given their presence in an experimentally tractable host, these potentially active elements might serve as useful models for the study of DIRS element retrotransposition. An element closely related to TcDirs1 was also detected in sequences from a second arthropod, the honey bee Apis mellifera (Hymenoptera), suggesting that these retrotransposons are long-term residents of arthropod genomes.

A group of deuterostome Ty3/ gypsy-like retrotransposons with Ty1/ copia-like pol-domain orders.

Here we report the existence of an unusual group of LTR retrotransposons, termed Gmr1-like elements. The members of this group are most similar in sequence to elements of the Ty3/ gypsy group, yet, unlike typical Ty3/ gypsy elements, they have pol genes in which the integrase domain lies upstream, rather than downstream, of the reverse transcriptase domain. Such an arrangement was formerly believed to be a characteristic peculiar to Ty1/ copia elements. The group includes the previously described retrotransposon Gmr1 from the Atlantic cod Gadus morhua, together with elements from a variety of other vertebrates, such as the zebrafish Danio rerio, the pufferfish Fugu rubripes and the clawed toad Xenopus laevis, as well as elements from non-vertebrate deuterostomes such as the sea squirt Ciona intestinalis. No Gmr1-like elements were found outside of the deuterostomes, nor were any other groups of elements with atypical pol-domain orders identified. Phylogenetic analyses show that the Gmr1-like elements form a monophyletic group within the larger group of Ty3/ gypsy elements. Some of the newly identified elements appear to be structurally intact and may still be active. The identification of this group challenges some previously held beliefs concerning the structure and evolution of LTR retrotransposons.

A nuclear-encoded intein in the fungal pathogen Cryptococcus neoformans.

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 DIRS1 group of retrotransposons.

Only three retrotransposons of the DIRS1 group have previously been described: DIRS1 from the slime mold Dictyostelium discoideum, PAT from the nematode Panagrellus redivivus, and Prt1 from the zygomycetous fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these elements suggest that they are related to the long terminal repeat (LTR) retroelements, such as the Ty3/gypsy retrotransposons and the vertebrate retroviruses. The DIRS1-group elements, however, have several unusual structural features which distinguish them from typical LTR elements: (1) they lack the capacity to encode DDE-type integrases or aspartic proteases; (2) they have open reading frames (ORFs) of unknown function; (3) they integrate without creating duplications of their target sites; and (4) although they are bordered by terminal repeats, these sequences differ from typical LTRs in that they are either inverted repeats or "split" direct repeats. Because of the small number of DIRS1-like elements described, and the unusual structures of these elements, little is known about their evolution, distribution, and replication mechanisms. Here, we report the identification of several new DIRS1-like retrotransposons, including elements from nematodes, sea urchins, fish, and amphibia. We also present evidence for the existence of DIRS1-like sequences in the human genome. In addition, we show that the lack of DDE-type integrase genes from elements of the DIRS1 group is explained by the finding that the previously uncharacterized ORFs of these elements encode proteins related to the site-specific recombinase of bacteriophage lambda. The presence of lambda-recombinase-like genes in DIRS1 elements also accounts for the lack of target-site duplications for these elements and may be related to the unusual structures of their terminal repeats.

An active retrotransposon in Candida albicans.

Tca2 is a Ty1/copia-type retrotransposon from the pathogenic yeast Candida albicans. It was originally identified as an abundant, linear, extrachromosomal, double-stranded DNA molecule. Here we show that Tca2 is widespread in C.albicans, but that the abundance of extrachromosomal Tca2 DNA varies greatly among different strains and is strongly dependent on the growth temperature. The relative levels of Tca2 RNA vary in a similar pattern to the extrachromosomal DNA, raising the possibility that the variations in extrachromosomal DNA levels are introduced predominantly at the level of transcription. We have also analysed the retrotranspositional activity of the element by developing a transposition assay involving a cloned Tca2 element tagged with a selectable marker gene that is activated by passage through an RNA intermediate. We show that the marked Tca2 is transpositionally active as transposed copies of the marked element became integrated at a variety of new positions in the genome and an intron in the donor element was precisely removed in the newly transposed copies. This is the first report of transposition in C.albicans.

L1-like non-LTR retrotransposons in the yeast Candida albicans.

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.

The diversity of retrotransposons in the yeast Cryptococcus neoformans.

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.

Current awareness on yeast.

In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals - search completed 4th Apr. 2001)

New BEL-like LTR-retrotransposons in Fugu rubripes, Caenorhabditis elegans, and Drosophila melanogaster.

The BEL group of retroelements is present in greater numbers, variety and taxonomic range than may have been thought previously. In addition to the insects, nematodes and schistosomes, BEL-like elements are present in echinoderms, urochordates, and at least two highly diverged species of fish. We describe one new full-length BEL-like element in Fugu that we call Suzu, another in Drosophila that we call Tinker, and seven new families in C. elegans. Many of the C. elegans elements have an unusual insertion at the 5' end. The previously known Roo, TRAM and Telemac are also BEL-like retrotransposons. Some BEL-like elements have captured an envelope gene, probably from other retroelements in some cases but from a phlebovirus in one case.

Tca5, a Ty5-like retrotransposon from Candida albicans.

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.

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans.

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.

The CARE-2 and rel-2 repetitive elements of Candida albicans contain LTR fragments of a new retrotransposon.

CARE-2 and Rel-2 are dispersed, repetitive elements of Candida albicans. Hybridisation experiments suggest that they are present at 10-20 copies per genome and appear on most, if not all, of the chromosomes. A high degree of interstrain variation has been demonstrated for CARE-2, making it of use for strain typing. Until now, however, the nature of the repetitive elements within CARE-2 and Rel-2 was unknown. We show here that CARE-2 and Rel-2 contain long terminal repeat (LTR) fragments of a new retrotransposon. These LTRs, which we designate kappa, are partially responsible for the repetitive nature of CARE-2 and Rel-2. Complete copies of the kappa elements are present elsewhere in the genome and adjacent to some are sequences characteristic of the internal regions of retrotransposons. An apparently high degree of scrambling of the kappa elements suggests that they may represent a hotspot for mutation and recombination in C. albicans.

pCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans.

Retrotransposons are mobile genetic elements. They can transpose via the reverse transcription of mRNA into double-stranded DNA (dsDNA) followed by the insertion of this dsDNA into new sites within the host genome. The unintegrated, linear, dsDNA form of retrotransposons is usually very rare. We report here the isolation of a retrotransposon from Candida albicans which is unusual in this respect. This element, which we have named pCal, was first identified as a distinct band when uncut C. albicans DNA was examined on an agarose gel. Sequence analysis of the cloned element revealed that it is a retrotransposon belonging to the Ty1/copia group. It is estimated that pCal produces 50 to 100 free, linear, dsDNA copies of itself per cell. This is a much higher level of expression than even that of the system in which Ty1 is expressed behind the highly active GAL1 promoter on a high-copy-number plasmid (about 10 copies per cell). Another unusual feature of pCal is that its Pol enzymes are likely to be expressed via the pseudoknot-assisted suppression of an upstream, in-phase stop codon, as has been shown for Moloney murine leukemia virus.

Interspecific complementation analysis by protoplast fusion of Candida tropicalis and Candida albicans adenine auxotrophs.

A protocol employing inositol starvation was used to isolate proline and adenine auxotrophs of Candida tropicalis. Interspecific hybrids between red adenine auxotrophs of C. tropicalis and Candida albicans were formed by protoplast fusion. These C. tropicalis red adenine auxotrophs were shown to fall into two complementation groups by crossing them with a known C. albicans ade1 tester strain. It is suggested that these two groups correspond to the ade1 and ade2 mutants of Saccharomyces cerevisiae and C. albicans and that these defined mutants may be useful in attempts to develop transformation systems for C. tropicalis.

Natural auxotrophic heterozygosity in Candida albicans.

Candida albicans is imperfect and diploid. This unusual genetic state permits the occurrence of strains heterozygous for recessive deleterious alleles. These alleles can be brought to homozygosity and therefore expression by UV irradiation-induced mitotic crossing over. Heterozygosity for recessive auxotrophic alleles is found in 10 to 15% of the strains. A wide variety of auxotrophic alleles have been found in such natural heterozygotes but one type, termed Suf +/- was found at an unusually high frequency. Some 5 to 10% of strains are of the Suf +/- type. Suf- homozygous auxotrophs are defective in sulfite reductase. Such auxotrophs require a source of reduced sulfur such as methionine or cysteine, alternatively inorganic thiosulfate will serve as a supplement. Complementation analysis of a variety of Suf- auxotrophs established that 11 out of 12 strains were in a single complementation group.

A Candida albicans mutant impaired in the utilization of N-acetylglucosamine.

Indicator plates containing eosin, methylene blue, glucosamine and proline were used to select mutants of Candida albicans impaired in the utilization of glucosamine. One such mutant, strain hOG298, grew on glucosamine at a slower rate than the parent and was severely impaired in growth on N-acetylglucosamine. The mutant was unable to express the first three steps in the N-acetylglucosamine pathway: viz the permease, N-acetylglucosamine kinase and N-acetylglucosamine-6-phosphate deacetylase. Glucosamine-6-phosphate deaminase was, however, induced by N-acetylglucosamine. The mutant still possessed a constitutive uptake system and kinase activity for glucosamine but glucosamine neither increased the glucosamine kinase activity nor induced N-acetylglucosamine kinase. These findings accounted for the decreased growth rate on glucosamine. The parent strain formed germ-tubes in N-acetylglucosamine or 4% (v/v) serum but the mutant formed germ-tubes only in serum.

Isolation and morphological characterization of a mycelial mutant of Candida albicans.

In this paper we describe the isolation of a novel strain of Candida albicans which is a mycelium at ambient temperatures. Mutagenesis of C. albicans ATCC 10261 with N-methyl-N-nitro-N-nitrosoguanidine followed by plating on solid media at 28 degrees C yielded colony morphology variants which were characterized by a raised, rough-surfaced colony of irregular outline in marked contrast to the flat, shiny circular colonies of the parental 10261 strain. One mutant colony, hOG301, was studied in detail. Strain hOG301 was stable and exhibited mycelial morphology over a wide temperature range (5 to 40 degrees C) in several media. The hyphae comprising hOG301 mycelium were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy and showed morphological features described in the literature as being typical of both true hyphae and pseudohyphae. In contrast to 10261, hOG301 was not pathogenic after intraperitoneal injection in mice. This is the first report of a mycelial C. albicans that is stable at ambient temperatures.

Characterization of a tetraploid derivative of Candida albicans ATCC 10261.

A morphometric analysis of Candida albicans yeast cells utilizing scanning electron microscopy showed that the cell volume and the DNA content of a tetraploid strain (derived by cell fusion) were 2.4 to 3.0 and 2.0 times, respectively, those of the progenitor diploid strain, ATCC 10261. The pathogenicities of both strains were similar.