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Molecular dissection of Penelope transposable element regulatory machinery.

Schostak N, Pyatkov K, Zelentsova E, Arkhipova I, Shagin D, Shagina I, Mudrik E, Blintsov A, Clark I, Finnegan DJ, Evgen'ev M - Nucleic Acids Res. (2008)

Bottom Line: Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown.The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass.The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology RAS, Moscow, Russia.

ABSTRACT
Penelope-like elements (PLEs) represent a new class of retroelements identified in more than 80 species belonging to at least 10 animal phyla. Penelope isolated from Drosophila virilis is the only known transpositionally active representative of this class. Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown. In order to determine the localization of presumptive Penelope promoter and enhancer-like elements, segments of the putative Penelope regulatory region were linked to a CAT reporter gene and introduced into D. melanogaster by P-element-mediated transformation. The results obtained using ELISA to measure CAT expression levels and RNA studies, including RT-PCR, suggest that the active Penelope transposon contains an internal promoter similar to the TATA-less promoters of LINEs. The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass. The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.

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(A) Histogram illustrating CAT levels in the ovaries and in carcasses of the transgenic strains. The schematic structure of all constructs used is depicted at the bottom. (B) Presumptive structure of the Penelope regulatory region with positions of promoter and other regulatory sequences indicated. Letters denote the constructs used, and CAT expression in ovaries (ov) and carcasses (ca) is illustrated by + or −. Figures at the bottom indicate the boundaries of constructs within the Penelope 5′ region (interval 1–850 bp). The dotted grey line shows promoter prediction scores from the NNPP program (from 0 to 1, indicated on the right). McPromoter scores yielded a qualitatively similar picture with different ratios of peak intensities (data not shown). The shaded region at the beginning of the ORF marks the 34-bp repeat.
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Figure 3: (A) Histogram illustrating CAT levels in the ovaries and in carcasses of the transgenic strains. The schematic structure of all constructs used is depicted at the bottom. (B) Presumptive structure of the Penelope regulatory region with positions of promoter and other regulatory sequences indicated. Letters denote the constructs used, and CAT expression in ovaries (ov) and carcasses (ca) is illustrated by + or −. Figures at the bottom indicate the boundaries of constructs within the Penelope 5′ region (interval 1–850 bp). The dotted grey line shows promoter prediction scores from the NNPP program (from 0 to 1, indicated on the right). McPromoter scores yielded a qualitatively similar picture with different ratios of peak intensities (data not shown). The shaded region at the beginning of the ORF marks the 34-bp repeat.

Mentions: We have previously shown that the p6 copy of D. virilis Penelope (Figure 1B) is functional in D. melanogaster and can actively transpose in transgenic strains (16). Moreover, the full-sized Penelope transcript is restricted to the ovaries of D. melanogaster strains transformed with an active Penelope copy, as is the case with dysgenic females of D. virilis. In order to show that the Penelope 5′ regulatory sequences are able to drive the expression of a reporter gene in the cells of the two species studied, we performed preliminary experiments which have shown that nucleotides 1–850 of this element (Figures 1B and 3B) are sufficient to drive expression of the chloramphenicol acetyl transferase (CAT) reporter gene upon transfection into D. virilis tissue culture cells (data not shown). Furthermore, we observed lacZ expression in the egg cyst and in the cytoplasm of the oocyte at different stages of oogenesis (Figure 2A–D) in the ovaries of transgenic D. melanogaster flies carrying the construct ‘PB’ (see Materials and Methods section) containing nucleotides 352–718 of Penelope linked to a lacZ reporter gene (11).Figure 2.


Molecular dissection of Penelope transposable element regulatory machinery.

Schostak N, Pyatkov K, Zelentsova E, Arkhipova I, Shagin D, Shagina I, Mudrik E, Blintsov A, Clark I, Finnegan DJ, Evgen'ev M - Nucleic Acids Res. (2008)

(A) Histogram illustrating CAT levels in the ovaries and in carcasses of the transgenic strains. The schematic structure of all constructs used is depicted at the bottom. (B) Presumptive structure of the Penelope regulatory region with positions of promoter and other regulatory sequences indicated. Letters denote the constructs used, and CAT expression in ovaries (ov) and carcasses (ca) is illustrated by + or −. Figures at the bottom indicate the boundaries of constructs within the Penelope 5′ region (interval 1–850 bp). The dotted grey line shows promoter prediction scores from the NNPP program (from 0 to 1, indicated on the right). McPromoter scores yielded a qualitatively similar picture with different ratios of peak intensities (data not shown). The shaded region at the beginning of the ORF marks the 34-bp repeat.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2377424&req=5

Figure 3: (A) Histogram illustrating CAT levels in the ovaries and in carcasses of the transgenic strains. The schematic structure of all constructs used is depicted at the bottom. (B) Presumptive structure of the Penelope regulatory region with positions of promoter and other regulatory sequences indicated. Letters denote the constructs used, and CAT expression in ovaries (ov) and carcasses (ca) is illustrated by + or −. Figures at the bottom indicate the boundaries of constructs within the Penelope 5′ region (interval 1–850 bp). The dotted grey line shows promoter prediction scores from the NNPP program (from 0 to 1, indicated on the right). McPromoter scores yielded a qualitatively similar picture with different ratios of peak intensities (data not shown). The shaded region at the beginning of the ORF marks the 34-bp repeat.
Mentions: We have previously shown that the p6 copy of D. virilis Penelope (Figure 1B) is functional in D. melanogaster and can actively transpose in transgenic strains (16). Moreover, the full-sized Penelope transcript is restricted to the ovaries of D. melanogaster strains transformed with an active Penelope copy, as is the case with dysgenic females of D. virilis. In order to show that the Penelope 5′ regulatory sequences are able to drive the expression of a reporter gene in the cells of the two species studied, we performed preliminary experiments which have shown that nucleotides 1–850 of this element (Figures 1B and 3B) are sufficient to drive expression of the chloramphenicol acetyl transferase (CAT) reporter gene upon transfection into D. virilis tissue culture cells (data not shown). Furthermore, we observed lacZ expression in the egg cyst and in the cytoplasm of the oocyte at different stages of oogenesis (Figure 2A–D) in the ovaries of transgenic D. melanogaster flies carrying the construct ‘PB’ (see Materials and Methods section) containing nucleotides 352–718 of Penelope linked to a lacZ reporter gene (11).Figure 2.

Bottom Line: Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown.The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass.The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology RAS, Moscow, Russia.

ABSTRACT
Penelope-like elements (PLEs) represent a new class of retroelements identified in more than 80 species belonging to at least 10 animal phyla. Penelope isolated from Drosophila virilis is the only known transpositionally active representative of this class. Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown. In order to determine the localization of presumptive Penelope promoter and enhancer-like elements, segments of the putative Penelope regulatory region were linked to a CAT reporter gene and introduced into D. melanogaster by P-element-mediated transformation. The results obtained using ELISA to measure CAT expression levels and RNA studies, including RT-PCR, suggest that the active Penelope transposon contains an internal promoter similar to the TATA-less promoters of LINEs. The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass. The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.

Show MeSH
Related in: MedlinePlus