EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.     

Cophylogeny and disparate rates of evolution in sympatric lineages of chewing lice on pocket gophers

Although molecular-based phylogenetic studies of hosts and parasites are increasingly common in the literature, no study to date has examined two congeneric lineages of parasites that live in sympatry on the same lineage of hosts. This study examines phylogenetic relationships among chewing lice (Phthiraptera: Trichodectidae) of the Geomydoecus coronadoi and Geomydoecus mexicanus species complexes and compares these to phylogenetic patterns in their hosts (pocket gophers of the rodent family Geomyidae). Sympatry of congeneric lice provides a natural experiment to test the hypothesis that closely related lineages of parasites will respond similarly to the same host. Sequence data from the mitochondrial COI and the nuclear EF-1alpha genes confirm that the two louse complexes are reciprocally monophyletic and that individual clades within each species complex parasitize a different species of pocket gopher. Phylogenetic comparisons reveal that both louse complexes show a significant pattern of cophylogeny with their hosts. Comparisons of rates of nucleotide substitution at 4-fold degenerate sites in the COI gene indicate that both groups of lice have significantly higher basal mutation rates than their hosts. The two groups of lice have similar basal rates of mutation, but lice of the G. coronadoi complex show significantly elevated rates of nucleotide substitution at all sites. These rate differences are hypothesized to result from population-level phenomena, such as effective population size, founder effects, and drift, that influence rates of nucleotide substitution.     

Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

The generality of habitat suitability models: A practical test with two insect groups

For the design and declaration of conservation areas as well as for planning habitat management it is important to quantitatively know the habitat preferences of the focal species. To take into account the requirements of as many species as possible, it would be of great advantage if one would either (i) find one or several species whose habitat requirements cover those of a large number of other species or if one could (ii) identify a common set of habitat parameters that is important for the occurrence of many species. Ideally such common habitat parameters should be easy to measure. Only then they may be of practical value in applied conservation biology.In this study, we compared the habitat preferences of different insect species (grasshoppers, bush crickets, butterflies, moths) in the same region by applying identical methods. To identify common explanatory variables that predict the occurrence probability of these species, we first tested the transferability of the specific ‘species models’ to other species within the same insect group. We tested how well the incidence of one species can be predicted by the occurrence probability of another species. The ‘best’ models within each group were then tested for transferability between the different groups. Additionally, we tested the predictive power of the predictor variable ‘habitat type’ as an easy and often available measure for conservation practice.Although in the different ‘species models’ different key factors determine habitat suitability, some models were successfully transferred and were able to reasonably predict the distribution of other species. The habitat preferences of the burnet moth Zygaena carniolica were particularly well suited for the prediction of suitable habitats for all other species. In addition, the predictor variable ‘habitat type’ played a dominant role in all models. Models using this aggregated predictor variable may well predict suitable habitat for all species.     

General and MicroRNA-Mediated mRNA Degradation Occurs on Ribosome Complexes in Drosophila Cells

The translation and degradation of mRNAs are two key steps in gene expression that are highly regulated and targeted by many factors, including microRNAs (miRNAs). While it is well established that translation and mRNA degradation are tightly coupled, it is still not entirely clear where in the cell mRNA degradation takes place. In this study, we investigated the possibility of mRNA degradation on the ribosome in Drosophila cells. Using polysome profiles and ribosome affinity purification, we could demonstrate the copurification of various deadenylation and decapping factors with ribosome complexes. Also, AGO1 and GW182, two key factors in the miRNA-mediated mRNA degradation pathway, were associated with ribosome complexes. Their copurification was dependent on intact mRNAs, suggesting the association of these factors with the mRNA rather than the ribosome itself. Furthermore, we isolated decapped mRNA degradation intermediates from ribosome complexes and performed high-throughput sequencing analysis. Interestingly, 93% of the decapped mRNA fragments (approximately 12,000) could be detected at the same relative abundance on ribosome complexes and in cell lysates. In summary, our findings strongly indicate the association of the majority of bulk mRNAs as well as mRNAs targeted by miRNAs with the ribosome during their degradation.