Performance of Forest Bryophytes with Different Geographical Distributions Transplanted across a Topographically Heterogeneous Landscape

Most species distribution models assume a close link between climatic conditions and species distributions. Yet, we know little about the link between species'' geographical distributions and the sensitivity of performance to local environmental factors. We studied the performance of three bryophyte species transplanted at south- and north-facing slopes in a boreal forest landscape in Sweden. At the same sites, we measured both air and ground temperature. We hypothesized that the two southerly distributed species Eurhynchium angustirete and Herzogiella seligeri perform better on south-facing slopes and in warm conditions, and that the northerly distributed species Barbilophozia lycopodioides perform better on north-facing slopes and in relatively cool conditions. The northern, but not the two southern species, showed the predicted relationship with slope aspect. However, the performance of one of the two southern species was still enhanced by warm temperatures. An important reason for the inconsistent results can be that microclimatic gradients across landscapes are complex and influenced by many climate-forcing factors. Therefore, comparing only north- and south-facing slopes might not capture the complexity of microclimatic gradients. Population growth rates and potential distributions are the integrated results of all vital rates. Still, the study of selected vital rates constitutes an important first step to understand the relationship between population growth rates and geographical distributions and is essential to better predict how climate change influences species distributions.     

Small RNAs of Rous Sarcoma Virus: Characterization by Two-Dimensional Polyacrylamide Gel Electrophoresis and Fingerprint Analysis

Approximately 15 to 20 different species of small (4 to 7S) RNAs have been purified by two-dimensional polyacrylamide gel electrophoresis of RNA isolated from virions of Schmidt-Ruppin D strain of Rous sarcoma virus. Each species of small RNA has been isolated free of 70S RNA; nine of them, including 5S and 7S RNAs, were also found associated with the 70S genomic RNA. Most of the 4S RNAs are present at an average of less than one copy per virion. The 4S RNAs have T1 RNase (EC 2.7.7.26) fingerprints, which are very similar to those of tRNAs. One of the smallest 4S RNAs, which can act as a primer for initiation of RNA-directed DNA synthesis, is associated with the 70S RNA in 1 to 2 copies per complex, whereas an additional 6 to 8 copies of this molecule are free.     

Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism

Background

Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine.

Results

We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome.

Conclusions

We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1090) contains supplementary material, which is available to authorized users.     

Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin

The three anomalously pigmented dinoflagellates Gymnodinium galatheanum, Gyrodinium aureolum, and Gymnodinium breve have plastids possessing 19'-hexanoyloxy-fucoxanthin as the major carotenoid rather than peridinin, which is characteristic of the majority of the dinoflagellates. Analyses of SSU rDNA from the plastid and the nuclear genome of these dinoflagellate species indicate that they have acquired their plastids via endosymbiosis of a haptophyte. The dinoflagellate plastid sequences appear to have undergone rapid sequence evolution, and there is considerable divergence between the three species. However, distance, parsimony, and maximum-likelihood phylogenetic analyses of plastid SSU rRNA gene sequences place the three species within the haptophyte clade. Pavlova gyrans is the most basal branching haptophyte and is the outgroup to a clade comprising the dinoflagellate sequences and those of other haptophytes. The haptophytes themselves are thought to have plastids of a secondary origin; hence, these dinoflagellates appear to have tertiary plastids. Both molecular and morphological data divide the plastids into two groups, where G. aureolum and G. breve have similar plastid morphology and G. galatheanum has plastids with distinctive features.     

Refining the Ciona intestinalis Model of Central Nervous System Regeneration

Background

New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism.

Methodology/Principal Findings

We developed techniques for reproducible ablations and for imaging live cellular events in tissue explants. Based on live observations of more than 100 regenerating animals, we subdivided the regeneration process into four stages. Regeneration was functional, as shown by the sequential recovery of reflexes that established new criteria for defining regeneration rates. We used transgenic animals and labeled nucleotide analogs to describe in detail the early cellular events at the tip of the regenerating nerves and the first appearance of the new adult ganglion anlage.

Conclusions/Significance

The rate of regeneration was found to be negatively correlated with adult size. New neural structures were derived from the anterior and posterior nerve endings. A blastemal structure was implicated in the formation of new neural cells. This work demonstrates that Ciona intestinalis is as a useful system for studies on regeneration of the brain, brain-associated organs and nerves.     

Nucleocytoplasmic transport and processing of small nuclear RNA precursors.

We have analyzed the structures and locations of small nuclear RNA (snRNA) precursors at various stages in their synthesis and maturation. In the nuclei of pulse-labeled Xenopus laevis oocytes, we detected snRNAs that were longer than their mature forms at their 3' ends by up to 10 nucleotides. Analysis of the 5' caps of these RNAs and pulse-chase experiments showed that these nuclear snRNAs were precursors of the cytoplasmic pre-snRNAs that have been observed in the past. Synthesis of pre-snRNAs was not abolished by wheat germ agglutinin, which inhibits export of the pre-snRNAs from the nucleus, indicating that synthesis of these RNAs is not obligatorily coupled to their export. Newly synthesized U1 RNAs could be exported from the nucleus regardless of the length of the 3' extension, but pre-U1 RNAs that were elongated at their 3' ends by more than about 10 nucleotides were poor substrates for trimming in the cytoplasm. The structure at the 3' end was critical for subsequent transport of the RNA back to the nucleus. This requirement ensures that truncated and incompletely processed U1 RNAs are excluded from the nucleus.     

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.     

Biochemical properties of the bromodeoxyuridine-induced guinea pig virus.

The biophysical and biochemical properties of the virus particles released by guinea pig embryo cells treated with 5-bromo-2'-deoxyuridine (BUdR) have been compared to those of the B-type mouse mammary tumor virus (MMTV) and the C-type Rauscher murine leukemia virus. The high-molecular-weight (60 to 70S) RNA of the BUdR-induced guinea pig virus (GPV) has a molecular weight of 8 X 106 when measred by mixed agarose polyacylamide gel electrophoresis. The virus particles isolated from the tissue culture medium of BUdR-induced guniea pig cells have the following properties in common with MMTV: (i) a buoyant density of 1.18 g/ml in sucrose and 1.21 g/ml in CsCl, and (ii) a DNA polymerase that prefers Mg2+ over Mn2+ in an assay using the synthetic template poly(rC):oligo(dG). No nucleic acid sequence homology between GPV RNA and the viral RNAs of the MMTV, murine leukemia virus, hamster sarcoma virus, or Mason-Pfizer monkey virus could be observed in a competition hybridization assay using the radioactive-labeled GPV 60 to 70S RNA. By this same competition by hybridization assay the frequency of GPV proviral sequences was estimated to be at least 83 per haploid cellular genome of guniea pig cells. No nucleic acid sequences related to be GPV RNA were detected in the DNA of normal tissues of mice, rats, cats, dogs, baboons, or humans by direct RNA-DNA hybridization using radioactive GPV60 to 70S RNA.