Telomerase in kinetoplastid parasitic protozoa

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major, and Leishmania tarentolae. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. (1994) Science 266, 2011–2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996) EMBO J. 15, 5928–5935]. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.

Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

Host–parasite relatedness shown by protein fingerprinting in a brood parasitic bird

Brood parasitism as an alternative female breeding tactic is particularly common in ducks, where hosts often receive eggs laid by parasitic females of the same species and raise their offspring. Herein, we test several aspects of a kin selection explanation for this phenomenon in goldeneye ducks (Bucephala clangula) by using techniques of egg albumen sampling and statistical bandsharing analysis based on resampling. We find that host and primary parasite are indeed often related, with mean r = 0.13, about as high as between first cousins. Relatedness to the host is higher in nests where a parasite lays several eggs than in those where she lays only one. Returning young females parasitize their birth nestmates (social mothers or sisters, which are usually also their genetic mothers and sisters) more often than expected by chance. Such adult relatives are also observed together in the field more often than expected and for longer periods than other females. Relatedness and kin discrimination, which can be achieved by recognition of birth nestmates, therefore play a role in these tactics and probably influence their success.

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa?

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp

Wolbachia are bacteria that live in the cells of various invertebrate species to which they cause a wide range of effects on physiology and reproduction. We investigated the effect of Wolbachia infection in the parasitic wasp, Asobara tabida Nees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected by Wolbachia. The removal of Wolbachia by antibiotic treatment had a totally unexpected effect—aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treated Asobara citri, a closely related species that does not harbor Wolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents) are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production and Wolbachia density in females. We conclude that Wolbachia is necessary for oogenesis in these A. tabida strains, and this association would seem to be the first example of a transition from facultative to obligatory symbiosis in arthropod–Wolbachia associations.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes.

We have explored the evolutionary history of the Apicomplexa and two related protistan phyla, Dinozoa and Ciliophora, by comparing the nucleotide sequences of small subunit ribosomal RNA genes. We conclude that the Plasmodium lineage, to which the malarial parasites belong, diverged from other apicomplexan lineages (piroplasmids and coccidians) several hundred million years ago, perhaps even before the Cambrian. The Plasmodium radiation, which gave rise to several species parasitic to humans, occurred approximately 129 million years ago; Plasmodium parasitism of humans has independently arisen several times. The origin of apicomplexans (Plasmodium), dinoflagellates, and ciliates may be > 1 billion years old, perhaps older than the three multicellular kingdoms of animals, plants, and fungi. Digenetic parasitism independently evolved several times in the Apicomplexa.

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

How caterpillar-damaged plants protect themselves by attracting parasitic wasps.

Parasitic and predatory arthropods often prevent plants from being severely damaged by killing herbivores as they feed on the plants. Recent studies show that a variety of plants, when injured by herbivores, emit chemical signals that guide natural enemies to the herbivores. It is unlikely that herbivore-damaged plants initiate the production of chemicals solely to attract parasitoids and predators. The signaling role probably evolved secondarily from plant responses that produce toxins and deterrents against herbivores and antibiotics against pathogens. To effectively function as signals for natural enemies, the emitted volatiles should be clearly distinguishable from background odors, specific for prey or host species that feed on the plant, and emitted at times when the natural enemies forage. Our studies on the phenomena of herbivore-induced emissions of volatiles in corn and cotton plants and studies conducted by others indicate that (i) the clarity of the volatile signals is high, as they are unique for herbivore damage, produced in relatively large amounts, and easily distinguishable from background odors; (ii) specificity is limited when different herbivores feed on the same plant species but high as far as odors emitted by different plant species and genotypes are concerned; (iii) the signals are timed so that they are mainly released during the daytime, when natural enemies tend to forage, and they wane slowly after herbivory stops.