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

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

Structural and functional characterization of giant plant Ogre-like retrotransposons

Ogre elements represent a distinct group of Ty3/gypsy LTR retrotransposons occurring in a range of dicot plants. They are characterized by two specific features ? presence of long extra open reading frame in 5´ untranslated region with unknown function and a non-coding sequence containing several stop codons separating protease and reverse transcriptase domains which was proposed to be removed by splicing. This thesis describes the functional analysis of intron splicing in Ogre retrotransposons. Further, it investigates additional coding information not only in Ogre retrotransposons but in the whole group of Ty3/gypsy retroelements

All to all dot-plot comparison of the pea satellite repeats.

<p>With exception of TR-11 and TR-19, the sequences of different satellite DNA families show no similarity. A fragment of long monomer of TR-19 shows high similarity to TR-11. FISH experiments revealed that all loci of TR-19 repeat contain also TR-11, but only some of TR-11 loci contain TR-19 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002777#pgen-1002777-g006" target="_blank">Figure 6</a> and data not shown), indicating that these two repeats should be considered as different families. Sequences used for dot-plot comparison are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002777#pgen.1002777.s001" target="_blank">Dataset S1</a>.</p

The CenH3-containing domains are fully colocalized with tubulin.

<p>A: Immunodetection of tubulin and CenH3-1 on two isolated metaphase chromosomes 3. Although isolated chromosomes never remained attached to microtubules they rarely exhibited weak tubulin signals which fully colocalized with CenH3-1. B–C: Detection of tubulin and CenH3-1 on chromosomes prepared using squash technique. The squash technique allowed some chromosomes to remain attached to microtubules. Whenever present, the remnants of mitotic spindle attached to chromosomes at all CenH3-containing domains on both metaphase (B) and anaphase (C) chromosomes. Bar = 5 µm.</p

Association of satellite DNA sequences with CenH3-containing domains.

<p>The figure shows results of simultaneous detection of different families of satellite DNA by FISH (green) and immunodection of CenH3-1 (red). A–C, F–L and O–R: All families of satellite DNA showing high ChiP enrichment were found to be colocalized with regions containing CenH3-1. D–E and M–N: The DNA families with no ChIP enrichment but present in primary constrictions were indeed found outside of the CenH3-containing domains. Colocalization of CenH3-1 and satellite DNA families on chromosome 3 is shown in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002777#pgen-1002777-g004" target="_blank">Figure 4</a>. Bar = 5 µm.</p

Organization and DNA sequence composition of CenH3-containing domains in chromosome 3.

<p>A–B: Primary constriction of chromosome 3 contains three functional centromere domains as defined by the presence of CenH3-1. Correlative fluorescence and scanning electron microscopy images of the same chromosome using FluoroNanogold showed that the three domains recognized with fluorescence (A, red signals) are composed of multiple foci from markers (bright spots) near the surface of the primary constriction (B, backscattered electron micrograph). C: Secondary electron micrograph image of the same chromosome. The primary constriction exhibits few chromomeres and a typical longitudinal orientation of fibrillar substructures, to which the CenH3 domains roughly correspond. The arrows mark the CenH3-1 containing regions. D–F: Detection of three different families of satellite DNA by FISH (green) combined with immunodection of CenH3-1 (red). Each of the functional centromere domains is composed of different family of satellite DNA; the domain closest to the long arm is composed of PisTR-B (D), the middle one of TR-1 (E) and the one closest to the short arm of TR-18 (F). Chromosomes were counterstained with DAPI (blue). Bar = 2 µm (A and D–F) or 0.2 µm (B–C).</p