Complete mitochondrial genome sequences of the Arctic Ocean codfishes <Emphasis Type="Italic">Arctogadus glacialis</Emphasis> and <Emphasis Type="Italic">Boreogadus saida</Emphasis> reveal oriL and tRNA gene duplications

We have determined the complete mitochondrial genome sequences of the codfishes Arctogadus glacialis and Boreogadus saida (Order Gadiformes, Family Gadidae). The 16,644 bp and 16,745 bp mtDNAs, respectively, contain the same set of 37 structural genes found in all vertebrates analyzed so far. The gene organization is conserved compared to other Gadidae species, but with one notable exception. B. saida contains heteroplasmic rearrangement-mediated duplications that include the origin of light-strand replication and nearby tRNA genes. Examination of the mitochondrial control region of A. glacialis, B. saida, and four additional representative Gadidae genera identified a highly variable domain containing tandem repeat motifs in A. glacialis. Mitogenomic phylogeny based on the complete mitochondrial genome sequence, the concatenated protein-coding genes, and the derived protein sequences strongly supports a sister taxa affiliation of A. glacialis and B. saida.     

Characterization of the self-splicing products of two complex Naegleria LSU rDNA group I introns containing homing endonuclease genes.

The two group I introns Nae.L1926 and Nmo.L2563, found at two different sites in nuclear LSU rRNA genes of Naegleria amoebo-flagellates, have been characterized in vitro. Their structural organization is related to that of the mobile Physarum intron Ppo.L1925 (PpLSU3) with ORFs extending the L1-loop of a typical group IC1 ribozyme. Nae.L1926, Nmo.L2563 and Ppo.L1925 RNAs all self-splice in vitro, generating ligated exons and full-length intron circles as well as internal processed excised intron RNAs. Formation of full-length intron circles is found to be a general feature in RNA processing of ORF-containing nuclear group I introns. Both Naegleria LSU rDNA introns contain a conserved polyadenylation signal at exactly the same position in the 3' end of the ORFs close to the internal processing sites, indicating an RNA polymerase II-like expression pathway of intron proteins in vivo. The intron proteins I-NaeI and I-NmoI encoded by Nae.L1926 and Nmo.L2563, respectively, correspond to His-Cys homing endonucleases of 148 and 175 amino acids. I-NaeI contains an additional sequence motif homologous to the unusual DNA binding motif of three antiparallel beta sheets found in the I-PpoI endonuclease, the product of the Ppo.L1925 intron ORF.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.     

Heterogeneous communities with lognormal species abundance distribution: Species-area curves and sustainability

Heterogeneous species abundance models are models in which the dynamics differ between species, described by variation among parameters defining the dynamics. Using a dynamic and heterogeneous species abundance model generating the lognormal species abundance distribution it is first shown that different degrees of heterogeneity may result in equivalent species abundance distributions. An alternative to Preston's canonical lognormal model is defined by assuming that reduction in resources, for example reduction in available area, increases the density regulation of each species. This leads to species-individual curves and species-area curves that are approximately linear in a double logarithmic plot. Preston's canonical parameter gamma varies little along these curves and takes values in the neighborhood of one. Quite remarkably, the curves, which define the sensitivity of the community to area reductions, are independent of the heterogeneity among species for this model. As a consequence, the curves can be estimated from a single sample from the community using the Poisson lognormal distribution. It is shown how to perform sensitivity analysis with respect to over-dispersion in sampling relative to the Poisson distribution as well as sampling intensity, that is, the fraction of the community sampled. The method is exemplified by analyzing three simulated data sets.     

A mitogenomic approach to the taxonomy of pollocks: <Emphasis Type="Italic">Theragra chalcogramma</Emphasis> and <Emphasis Type="Italic">T. finnmarchica</Emphasis> represent one single species

Background  

The walleye pollock (Theragra chalcogramma) and Norwegian pollock (T. finnmarchica) are confined to the North Pacific and North Atlantic Oceans, respectively, and considered as distinct species within the family Gadidae. We have determined the complete mtDNA nucleotide sequence of two specimens of Norwegian pollock and compared the sequences to that of 10 specimens of walleye pollock representing stocks from the Sea of Japan and the Bering Sea, 2 specimens of Atlantic cod (Gadus morhua), and 2 specimens of haddock (Melanogrammus aeglefinus).     

Heterogeneity in dynamic species abundance models: the selective effect of extinction processes

There is often large variation in traits across the species of a community. In particular, variation in life history traits affecting population dynamics is likely to affect the species abundance distribution. Applying a dynamic and heterogeneous species abundance model we study how differences in extinction time for species in a community act as a force changing the distribution of dynamic parameters across species. This process may generate communities that are more heterogeneous then the heterogeneity measured as the species enter the community. Analytical results for some versions of the lognormal and gamma species abundance model are given as exemplifications of this process, together with stochastic simulations demonstrating the temporal changes in number of species and community heterogeneity through time.     

Contrasting patterns of density‐dependent selection at different life stages can create more than one fast–slow axis of life‐history variation

There has been much recent research interest in the existence of a major axis of life‐history variation along a fast–slow continuum within almost all major taxonomic groups. Eco‐evolutionary models of density‐dependent selection provide a general explanation for such observations of interspecific variation in the "pace of life." One issue, however, is that some large‐bodied long‐lived “slow” species (e.g., trees and large fish) often show an explosive “fast” type of reproduction with many small offspring, and species with “fast” adult life stages can have comparatively “slow” offspring life stages (e.g., mayflies). We attempt to explain such life‐history evolution using the same eco‐evolutionary modeling approach but with two life stages, separating adult reproductive strategies from offspring survival strategies. When the population dynamics in the two life stages are closely linked and affect each other, density‐dependent selection occurs in parallel on both reproduction and survival, producing the usual one‐dimensional fast–slow continuum (e.g., houseflies to blue whales). However, strong density dependence at either the adult reproduction or offspring survival life stage creates quasi‐independent population dynamics, allowing fast‐type reproduction alongside slow‐type survival (e.g., trees and large fish), or the perhaps rarer slow‐type reproduction alongside fast‐type survival (e.g., mayflies—short‐lived adults producing few long‐lived offspring). Therefore, most types of species life histories in nature can potentially be explained via the eco‐evolutionary consequences of density‐dependent selection given the possible separation of demographic effects at different life stages.     

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.