Stretching the rules: monocentric chromosomes with multiple centromere domains.

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

Differential response of Drosophila cell lines to extracellular adenosine.

Adenosine (Ado) is a crucial metabolite that affects a wide range of physiological processes. Key proteins regulating Ado signaling, transport and metabolism are conserved among vertebrates and invertebrates. It is well known that Ado influences proliferation of several vertebrate and invertebrate cells. Here we show that Ado negatively influences viability, changes morphology and mitochondrial polarity of the Drosophila imaginal disc cell line (Cl.8+) via a mechanism exclusively dependent on cellular Ado uptake. High transport of Ado is followed by phosphorylation and ATP production as a part of Ado salvation, which at higher concentrations may interfere with cellular homeostasis. In contrast, hematopoietic cell line Mbn2, which grows well in high Ado concentration, preferentially uses adenosine deaminase as a part of the purine catabolic pathway. Our results show that different types of Drosophila cell lines use different pathways for Ado conversion and suggest that such differences may be an important part of complex mechanisms maintaining energy homeostasis in the body.

A widespread occurrence of extra open reading frames in plant Ty3/gypsy retrotransposons.

Long terminal repeat (LTR) retrotransposons make up substantial parts of most higher plant genomes where they accumulate due to their replicative mode of transposition. Although the transposition is facilitated by proteins encoded within the gag-pol region which is common to all autonomous elements, some LTR retrotransposons were found to potentially carry an additional protein coding capacity represented by extra open reading frames located upstream or downstream of gag-pol. In this study, we performed a comprehensive in silico survey and comparative analysis of these extra open reading frames (ORFs) in the group of Ty3/gypsy LTR retrotransposons as the first step towards our understanding of their origin and function. We found that extra ORFs occur in all three major lineages of plant Ty3/gypsy elements, being the most frequent in the Tat lineage where most (77 %) of identified elements contained extra ORFs. This lineage was also characterized by the highest diversity of extra ORF arrangement (position and orientation) within the elements. On the other hand, all of these ORFs could be classified into only two broad groups based on their mutual similarities or the presence of short conserved motifs in their inferred protein sequences. In the Athila lineage, the extra ORFs were confined to the element 3' regions but they displayed much higher sequence diversity compared to those found in Tat. In the lineage of Chromoviruses the extra ORFs were relatively rare, occurring only in 5' regions of a group of elements present in a single plant family (Poaceae). In all three lineages, most extra ORFs lacked sequence similarities to characterized gene sequences or functional protein domains, except for two Athila-like elements with similarities to LOGL4 gene and part of the Chromoviruses extra ORFs that displayed partial similarity to histone H3 gene. Thus, in these cases the extra ORFs most likely originated by transduction or recombination of cellular gene sequences. In addition, the protein domain which is otherwise associated with DNA transposons have been detected in part of the Tat-like extra ORFs, pointing to their origin from an insertion event of a mobile element.

Experimental evidence for splicing of intron-containing transcripts of plant LTR retrotransposon Ogre.

Ogre elements are a distinct group of plant Ty3/gypsy-like retrotransposons characterized by several specific features, one of which is a separation of the gag-pol region into two non-overlapping open reading frames: ORF2 coding for Gag-Pro, and ORF3 coding for RT/RH-INT proteins. Previous characterization of Ogre elements from several plant species revealed that part of their transcripts lacks the region between ORF2 and ORF3, carrying one uninterrupted ORF instead. In this work, we investigated a hypothesis that this region represents an intron that is spliced out from part of the Ogre transcripts as a means for preferential production of ORF2-encoded proteins over those encoded by the complete ORF2-ORF3 region. The experiments involved analysis of transcription patterns of well-defined Ogre populations in a model plant Medicago truncatula and examination of transcripts carrying dissected pea Ogre intron expressed within a coding sequence of chimeric reporter gene. Both experimental approaches proved that the region between ORF2 and ORF3 is spliced from Ogre transcripts and showed that this process is only partial, probably due to weak splice signals. This is one of very few known cases of spliced LTR retrotransposons and the only one where splicing does not involve parts of the element's coding sequences, thus resembling intron splicing found in most cellular genes.