Similar patterns of rDNA evolution in synthetic and recently formed natural populations of <Emphasis Type="Italic">Tragopogon</Emphasis>(Asteraceae) allotetraploids

Background  

Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids.     

Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family

Background  

In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in ~200 species representing the family diversity and other closely related groups.     

Antimalarial Exposure Delays Plasmodium falciparum Intra-Erythrocytic Cycle and Drives Drug Transporter Genes Expression

Background

Multi-drug resistant Plasmodium falciparum is a major obstacle to malaria control and is emerging as a complex phenomenon. Mechanisms of drug evasion based on the intracellular extrusion of the drug and/or modification of target proteins have been described. However, cellular mechanisms related with metabolic activity have also been seen in eukaryotic systems, e.g. cancer cells. Recent observations suggest that such mechanism may occur in P. falciparum.

Methodology/Principal Findings

We therefore investigated the effect of mefloquine exposure on the cell cycle of three P. falciparum clones (3D7, FCB, W2) with different drug susceptibilities, while investigating in parallel the expression of four genes coding for confirmed and putative drug transporters (pfcrt, pfmdr1, pfmrp1 and pfmrp2). Mefloquine induced a previously not described dose and clone dependent delay in the intra-erythrocytic cycle of the parasite. Drug impact on cell cycle progression and gene expression was then merged using a non-linear regression model to determine specific drug driven expression. This revealed a mild, but significant, mefloquine driven gene induction up to 1.5 fold.

Conclusions/Significance

Both cell cycle delay and induced gene expression represent potentially important mechanisms for parasites to escape the effect of the antimalarial drug.     

Coupling between vesicle shape and the non-homogeneous lateral distribution of membrane constituents in Golgi bodies

In this work, a hypothesis is presented that could explain the non-homogeneous lateral distribution of membrane components in Golgi vesicles. It is shown that the non-homogeneous lateral distribution of membrane components and the specific flattened shape of Golgi vesicles are strongly coupled. In agreement with experimental evidence, it is indicated that some of the membrane components may be concentrated mainly on the curved bulbous rims of the Golgi vesicles, while the other components are distributed predominantly in their flat central part.     

Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana

An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.     

Endovesicle formation and membrane perturbation induced by polyoxyethyleneglycolalkylethers in human erythrocytes

Polyoxyethyleneglycolalkylether (CmEn, m=12, n=8) can induce a large torocyte-like endovesicle in human erythrocytes. The present study aimed to examine how variations in the molecular structure of CmEn (m=10,12,14,16,18; n=1-10,23) affect the occurrence of torocyte endovesicles. Our results show that torocytes occur most frequently when m=12,14 and n=8,9. At this molecular configuration the detergents induce inward membrane bending (stomatocytic S1-S2 shapes) resulting in the formation of a large membrane invagination. These detergents have a strong membrane perturbing, i.e., haemolytic, effect. Theoretical calculations indicate that a torocyte-shaped inside-out membrane vesicle can be created from a large membrane invagination due to the impact of laterally mobile anisotropic membrane inclusions. Such inclusions may be detergent-membrane component complexes or unanchored integral membrane proteins. It is shown that a nonhomogeneous lateral distribution of anisotropic membrane inclusions may stabilise the torocyte endovesicle shape, characterised by having opposite membranes in the thin central region of the vesicles separated by a certain distance. Tubular, conical or inverted conical isotropic inclusions cannot do so.     

The reverse tetracycline-controlled transactivator rtTA2s-S2 is toxic in mouse embryonic stem cells

The efficient and reversible control of transgene expression is a powerful tool for the correct manipulation of embryonic stem cells in both cell therapy and transgenesis. The aim of this work was to investigate the possibilities of recently developed reverse tetracycline-controlled transactivator rtTA2s-S2. We show that the rtTA2s-S2 is useful for transient inducible expression of genes in embryonic stem cells. However, we found that it was not possible to establish mouse embryonic stem cell lines stably expressing this transactivator. Using the viral IRES sequence which couples the expression of rtTA2s-S2 and neomycin phosphotransferase, we found that embryonic stem cells expressing rtTA2s-S2 are not capable of growing in the presence of G418. Our results indicate that this transactivator is toxic to ES cells and raise the need for the development of other strategies for stable and inducible expression of genes in ES cells.