DeviaTE: Assembly‐free analysis and visualization of mobile genetic element composition

Transposable elements (TEs) are selfish DNA sequences that multiply within host genomes. They are present in most species investigated so far at varying degrees of abundance and sequence diversity. The TE composition may not only vary between but also within species and could have important biological implications. Variation in prevalence among populations may for example indicate a recent TE invasion, whereas sequence variation could indicate the presence of hyperactive or inactive forms. Gaining unbiased estimates of TE composition is thus vital for understanding the evolutionary dynamics of transposons. To this end, we developed DeviaTE, a tool to analyse and visualize TE abundance using Illumina or Sanger sequencing reads. Our tool requires sequencing reads of one or more samples (tissue, individual or population) and consensus sequences of TEs. It generates a table and a visual representation of TE composition. This allows for an intuitive assessment of coverage, sequence divergence, segregating SNPs and indels, as well as the presence of internal and terminal deletions. By contrasting the coverage between TEs and single copy genes, DeviaTE derives unbiased estimates of TE abundance. We show that naive approaches, which do not consider regions spanned by internal deletions, may substantially underestimate TE abundance. Using published data we demonstrate that DeviaTE can be used to study the TE composition within samples, identify clinal variation in TEs, compare TE diversity among species, and monitor TE invasions. Finally we present careful validations with publicly available and simulated data. DeviaTE is implemented in Python and distributed under the GPLv3 ( https://github.com/W-L/deviaTE ).     

Resveratrol causes arrest in the S-phase prior to Fas-independent apoptosis in CEM-C7H2 acute leukemia cells

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), in the concentration range of 20 microM and above, induced arrest in the S-phase and apoptosis in the T cell-derived T-ALL lymphocytic leukemia cell line CEM-C7H2 which is deficient in functional p53 and p16. Expression of transgenic p16/INK4A, which causes arrest in G0/G1, markedly reduced the percentage of apoptotic cells. Antagonist antibodies to Fas or FasL, or constitutive expression of crmA did not diminish the extent of resveratrol-induced apoptosis. Furthermore, a caspase-8-negative, Fas-resistant Jurkat cell line was sensitive to resveratrol-induced apoptosis which could be strongly inhibited in the Jurkat as well as in the CEM cell line by z-VAD-fmk and z-IETD-fmk. The almost complete inhibition by z-IETD-fmk and the lack of inhibition by crmA suggested caspase-6 to be the essential initiator caspase. Western blots revealed the massive conversion of procaspase-6 to its active form, while caspase-3 and caspase-2 were proteolytically activated to a much lesser extent.     

Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo

Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E. The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)-dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.     

Spontaneous mutations restore the viability of tick-borne encephalitis virus mutants with large deletions in protein C

The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these "double mutants" provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.     

The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.     

Molecular characterization of the human protein kinase C θ * gene locus (PRKCQ)

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCθ, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCθ was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCθ cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16?000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated.     

Cell morphology, ultrastructure, and calcification pattern of Oocardium stratum, a peculiar lotic desmid

Cell morphology and ultrastructure of the desmid Oocardium stratum and its habitat conditions in two limestone-precipitating spring habitats in the Alps were studied. In spite of specific cell geometry, we found ultrastructural features (nucleus with nucleolus, Golgi apparatus, chloroplast structure, lipid bodies, cell wall texture) closely related to other desmids. The type of the mucilage pore apparatus perforating in high densities extended areas of the cell wall of Oocardium is of the Cosmarium type. Oocardium contrasts to Cosmarium by a peculiar bilateral cell geometry (lateral sphenoid shape) which is combined with a dislocated nucleus. Although the cell features of Oocardium did not differ between the two habitats, different calcification types (rhombohedral calcite versus fascicular-fibrous calcite) and calcification intensities were recorded. The spatial positioning and extension of the Oocardium niches differed considerably between the two springs in spite of high CO₂ oversaturation at both sites.