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. 2015 Sep 23;10(9):e0137050.
doi: 10.1371/journal.pone.0137050. eCollection 2015.

Achilles, a New Family of Transcriptionally Active Retrotransposons From the Olive Fruit Fly, With Y Chromosome Preferential Distribution

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Free PMC article

Achilles, a New Family of Transcriptionally Active Retrotransposons From the Olive Fruit Fly, With Y Chromosome Preferential Distribution

Konstantina T Tsoumani et al. PLoS One. .
Free PMC article

Abstract

Sex chromosomes have many unusual features relative to autosomes. The in depth exploration of their structure will improve our understanding of their origin and divergence (degeneration) as well as the evolution of genetic sex determination pathways which, most often are attributed to them. In Tephritids, the structure of Y chromosome, where the male-determining factor M is localized, is largely unexplored and limited data concerning its sequence content and evolution are available. In order to get insight into the structure and organization of the Y chromosome of the major olive insect pest, the olive fly Bactrocera oleae, we characterized sequences from a Pulse Field Gel Electrophoresis (PFGE)-isolated Y chromosome. Here, we report the discovery of the first olive fly LTR retrotransposon with increased presence on the Y chromosome. The element belongs to the BEL-Pao superfamily, however, its sequence comparison with the other members of the superfamily suggests that it constitutes a new family that we termed Achilles. Its ~7.5 kb sequence consists of the 5'LTR, the 5'non-coding sequence and the open reading frame (ORF), which encodes the polyprotein Gag-Pol. In situ hybridization to the B. oleae polytene chromosomes showed that Achilles is distributed in discrete bands dispersed on all five autosomes, in all centromeric regions and in the granular heterochromatic network corresponding to the mitotic sex chromosomes. The between sexes comparison revealed a variation in Achilles copy number, with male flies possessing 5-10 copies more than female (CI range: 18-38 and 12-33 copies respectively per genome). The examination of its transcriptional activity demonstrated the presence of at least one intact active copy in the genome, showing a differential level of expression between sexes as well as during embryonic development. The higher expression was detected in male germline tissues (testes). Moreover, the presence of Achilles-like elements in different species of the Tephritidae family suggests an ancient origin of this element.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cytogenetic distribution of partially Y-enriched libraries with preference towards the male DNA.
In situ hybridization to B. oleae a) metaphase & b, c) polytene chromosomes revealed that the element is dispersed throughout the genome and also located at heterochromatic regions.
Fig 2
Fig 2. Organization and structure of the putative intact Achilles element.
a) ORF1 encodes a Gag-Pol polyprotein consisting of 1632 aa. b) Conceptual translation of the Achilles coding region. The highly conserved amino acids are denoted by bold letters and highlighted in grey. The underlined amino acids and/or the above of them arrows represent the identifiable motifs shared with other retrotransposons. Their designations are shown on the right. The position marked with the red triangle (▽) indicates the deletion of the RH region. Asterisks (*) refer to the stop mutations within the coding sequences.
Fig 3
Fig 3. Phylogenetic analysis of Achilles.
The RT domains among representative members of the BEL/Pao family (MAX, GATE, BEL, Pao, ninja) were aligned with the corresponding region of Achilles using the ClustalW. The N-J tree was generated using as outgroup the copia element. Achilles is more related to the BEL elements. The numbers at the nodes are the bootstrap values based on 1000 pseudoreplications.
Fig 4
Fig 4. In situ hybridization of a digoxigenin-labelled Achilles-probe on B. oleae polytene chromosomes.
a) Polytene nucleus, b) part of polytene chromosome III, c) part of polytene chromosome IV. Spread preparations of polytene chromosomes were made from the salivary glands of third instar larvae as described by Drosopoulou et al. (1995). Centromeres are indicated by “c” and the corresponding chromosomes are demonstrated by their number (I-V). Multiple heterochromatic signals are indicated by multiple arrows (in Panel a). Discrete euchromatic signals are indicated by the number of the respective chromosomal division (Panels a, b, c).
Fig 5
Fig 5. Species distribution of Achilles.
Genomic PCR amplfication of Achilles-RT in in the 7 diptera species tested using AchillF2/R2 primer set. [Dm: Drosophila melanogaster, As: Anastrepha serpentina, Ast: Anastrepha striata, Cc: Ceratitis capitata, Bo: Bactrocera oleae, Bc: Bactrocera correcta and Bd: Bactrocera dorsalis.]
Fig 6
Fig 6. Expression profile of Achilles in specific tissues of both sexes as well as in embryos at various developmental stages.
Expression levels were detected by relative qRT-PCR in a) individual eggs collected at different time points during embryonic development and b) in two different tissues per gender of 7th day adult virgin flies: total bodies of females (Adult_F) and males (Adults_M) and the germline tissues testes and ovaries. Standard error of the mean of two biological replicates per time point is depicted in bars.

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Grant support

This work was partially supported by a grant from the Hellenic General Secretariat of Research and Technology (99 ED529), the Pythagoras II grant from the Greek Ministry of National Education and Religious Affairs and the two Graduate Programs of the Biochemistry and Biotechnology Department of the University of Thessaly (‘Biotechnology - Nutrition and Environment’ and ‘Molecular Biology and Genetics applications’). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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