Margaret I. Butler

A retrotransposon family from the pufferfish (fugu) Fugu rubripes

In this study we describe the isolation and characterisation of the first full-length vertebrate retrotransposon. Knowledge of vertebrate gypsy LTR-retrotransposons has been limited to short internal sequences from three fish and a corrupt sequence from a salamander. This paper describes the sequence of a full-length (5.645 kb) retrotransposon from the fugu fish Fugu rubripes. The retrotransposon, termed sushi-ichi (032H04), is a representative of a retrotransposon family (sushi) found as multiple copies within the fish genome. Two long open reading frames (ORFs) are predicted from the sequence. The first has homology to retroviral gag genes. The second includes sequences homologous to protease, reverse transcriptase/RNase H and integrase domains, in that order. Sequence comparisons of the predicted ORFs indicate that this element is related to the gypsy class of LTR-retrotransposons. Specifically, the sushi retrotransposons are most closely related to the retrotransposon group which includes the MAGGY retroelement from the rice blast fungus Magnaporthe grisea and the CfT-1 element from the fungal tomato pathogen Cladosporium fulvum.

Vertebrate LTR Retrotransposons of the Tf1/Sushi Group

Abstract. LTR retrotransposons of the Tf1/sushi group from a diversity of vertebrates, including fish, amphibians, and mammals (humans, mice, and others), are described as full-length or partial elements. These elements are compared, and the mechanisms involved in self-priming of reverse transcriptase and programmed phase shifting are inferred. Evidence is presented that in mammals these elements are still transcriptionally active and are represented as proteins. This suggests that members of the Tf1/sushi group are present as functional elements (or incorporated as partial elements into host genes) in diverse vertebrate lineages.

The distribution and evolutionary history of the PRP8 intein.

BackgroundWe 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.ResultsIn 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.ConclusionPRP8 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.

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 Cne PRP8 coding sequence and the flanking sequences of the Cryptococcus neoformans PRP8 gene have been assigned GenBank Accession No. AF349436. Copyright

Invertebrates, macroalgae, and chemical features in morainic ponds near Lakes Tekapo and Ohau, including new distribution records of Crustacea

Abstract The invertebrates and macroalgae in 42 permanent and temporary ponds and kettles in glacial moraine south of Lake Ohau, and 36 sites in moraine west of Lake Tekapo, South Island, New Zealand, were sampled in December 1980, and taxonomic lists were compiled. Most organisms belonged to taxa characteristically found in shallow waters elsewhere in New Zealand, but new distributions for the rare cladocerans Neothrix armata, Monospilus dispar, and Ephemeroporus barroisi were recorded, and the presence of Simocephalus acutirostratus in New Zealand was confirmed. Darkly pigmented Daphnia carinata were recorded for the first time as also were co‐occurring Boeckella triarticulata and Boeckella dilatata. Cluster analysis of the data from both areas identified 3 groups of taxa with a high probability of co‐occurrence. Some species were absent from all the sites in 1 area but present at several sites in the other. This may reflect differences in adaptation to drought, or the limited sampling regime, but is al...

Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

BackgroundInteins 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.ResultsWe 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.ConclusionThe 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.

Water Mite Predation on Planktonic Cladocera: Parallel Curve Analysis of Functional Responses

A water mite, Piona exigua, is the dominant invertebrate predator in some New Zealand lakes. To establish the extent to which this predator has an impact on the populations of its prey species, we have analysed the effect of prey availability on quantitative estimates of predation. We describe Piona functional responses generated under a range of conditions and compare them using «parallel curve analysis» and a maximum likelihood method of estimating curve parameters

Two new fungal inteins

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. Copyright

Prey selectivity of Piona exigua, a planktonic water mite

SummaryFemales, males and nymphs of Piona exigua were observed during prey capture and ingestion. The encounter radius of the mite was very small, allowing the escape of some crustaceans, such as the calanoid copepod Boeckella. Cladocerans, such as Bosmina or Chydorus, with little or no pre-contact escape response were the most vulnerable to mite predation. Preference values in size-selection experiments varied widely between individual mites. Adult mites presented with two sizes of Daphnia carinata generally preferred the smaller prey. When four sizes were presented simultaneously, however, the preferences of female mites for each size were not significantly different. Patterns of prey selection varied with predator age and sex; for example, female mites preferred Daphnia to Simocephalus, Ceriodaphnia and Chydorus, while nymphs showed a strong preference for Chydorus over Ceriodaphnia. When two prey types were present in equal proportions, differences in total prey density (range 5 or 10/1 – 30 or 50/1) did not alter preferences between the prey species. The preference of female mites for a particular prey type generally increased with increasing relative abundance of the prey type in each of three experiments (Daphnia: Ceriodaphnia, Ceriodaphnia: Chydorus, and Daphnia: Simocephalus). These results imply switching behaviour in these mites. Our results indicate the value of direct observation of predatory behaviour as an adjunct to prey selection experiments. It is also apparent that predatory behaviour in the presence of more than one prey type may not be predictable from that observed in single-prey situations. Predation rates on particular prey species were sometimes reduced in the presence of another species. The relative proportions of prey eaten when two species were present could not be predicted from the number of each species eaten when they were presented separately.

Ty3/gypsy-like retrotransposons in Candida albicans and Candida dubliniensis: Tca3 and Tcd3

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. Copyright