Limits...
Embryonic expression of endogenous retroviral RNAs in somatic tissues adjacent to the Oikopleura germline.

Henriet S, Sumic S, Doufoundou-Guilengui C, Jensen MF, Grandmougin C, Fal K, Thompson E, Volff JN, Chourrout D - Nucleic Acids Res. (2015)

Bottom Line: Tor Env proteins are membrane-associated glycoproteins which exhibit some features of viral membrane fusion proteins.Such embryonic expression depends on determinants present in the Tor elements and not on their surrounding genomic environment.Our study shows that unusual modes of transcription and expression close to the germline may contribute to the proliferation of Tor elements.

View Article: PubMed Central - PubMed

Affiliation: Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, N-5008, Norway simon.henriet@sars.uib.no.

Show MeSH

Related in: MedlinePlus

Autonomous expression of Torgenes. (A) Schematic representations of the expression constructs tested in Oikopleuraembryos. The numbers indicate coordinates on TorDNA, striped boxes represent non-coding sequences. (B) pCTor3b-2 drives Env expression in the anterior cells of the notochord. (BA), embryo before hatching; (BB) and (BC), embryos after hatching showing a complete notochord with 20 cells (blue arrows); (B’) and (B’’)), comparison of pCTor3b-2 activity with the expression pattern of Tor3b-2 env in wild-type embryos. (C) pCTor4b-1 expresses Env in various tissues. The table indicates the number of positive embryos showing expression in the same tissue (Supplementary Figure S7A). (D) Tor4b-1 copies and their envexpression pattern. The table shows the presence of insertions i1 to i14 in F1 animals from individual crosses. Segregation in the progeny was followed using Southern blotting (Supplementary Figure S7B). On the right, micrographs show WISH patterns obtained in the progeny using the antisense Tor4b-1 env probe. Numbers indicate signal frequency.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402516&req=5

Figure 6: Autonomous expression of Torgenes. (A) Schematic representations of the expression constructs tested in Oikopleuraembryos. The numbers indicate coordinates on TorDNA, striped boxes represent non-coding sequences. (B) pCTor3b-2 drives Env expression in the anterior cells of the notochord. (BA), embryo before hatching; (BB) and (BC), embryos after hatching showing a complete notochord with 20 cells (blue arrows); (B’) and (B’’)), comparison of pCTor3b-2 activity with the expression pattern of Tor3b-2 env in wild-type embryos. (C) pCTor4b-1 expresses Env in various tissues. The table indicates the number of positive embryos showing expression in the same tissue (Supplementary Figure S7A). (D) Tor4b-1 copies and their envexpression pattern. The table shows the presence of insertions i1 to i14 in F1 animals from individual crosses. Segregation in the progeny was followed using Southern blotting (Supplementary Figure S7B). On the right, micrographs show WISH patterns obtained in the progeny using the antisense Tor4b-1 env probe. Numbers indicate signal frequency.

Mentions: For the synthesis of RNA probes (Supplementary Figure S1), we amplified gene fragments with polymerase chain reaction (PCR) using specific primers and cDNA libraries from mixed developmental stages (1–6 h pf). The PCR products were cloned in pCRII-TOPO (Invitrogen) or pGEM-T vectors (Promega), prior to linearization for in vitro transcription with T7 or SP6 RNA polymerases (Roche). For expression in HEK293T cells, we cloned the complete env ORFs in C-terminal fusions with V5/6His tag in pCDNA 3.1-TOPO vector (Invitrogen). To construct pCTor3b-2 and pCTor4b-1, we generated DNA fragments containing part of env, fused with the V5/6His tag, followed by a stop codon and a restriction site (Figure 6A). These were then digested with the appropriate enzymes and ligated to upstream and downstream DNA fragments in order to reconstruct a Tor element that carried the full gag to the 3′LTR sequence. The resulting inserts were PCR amplified and cloned into the pCRII-TOPO vector.


Embryonic expression of endogenous retroviral RNAs in somatic tissues adjacent to the Oikopleura germline.

Henriet S, Sumic S, Doufoundou-Guilengui C, Jensen MF, Grandmougin C, Fal K, Thompson E, Volff JN, Chourrout D - Nucleic Acids Res. (2015)

Autonomous expression of Torgenes. (A) Schematic representations of the expression constructs tested in Oikopleuraembryos. The numbers indicate coordinates on TorDNA, striped boxes represent non-coding sequences. (B) pCTor3b-2 drives Env expression in the anterior cells of the notochord. (BA), embryo before hatching; (BB) and (BC), embryos after hatching showing a complete notochord with 20 cells (blue arrows); (B’) and (B’’)), comparison of pCTor3b-2 activity with the expression pattern of Tor3b-2 env in wild-type embryos. (C) pCTor4b-1 expresses Env in various tissues. The table indicates the number of positive embryos showing expression in the same tissue (Supplementary Figure S7A). (D) Tor4b-1 copies and their envexpression pattern. The table shows the presence of insertions i1 to i14 in F1 animals from individual crosses. Segregation in the progeny was followed using Southern blotting (Supplementary Figure S7B). On the right, micrographs show WISH patterns obtained in the progeny using the antisense Tor4b-1 env probe. Numbers indicate signal frequency.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402516&req=5

Figure 6: Autonomous expression of Torgenes. (A) Schematic representations of the expression constructs tested in Oikopleuraembryos. The numbers indicate coordinates on TorDNA, striped boxes represent non-coding sequences. (B) pCTor3b-2 drives Env expression in the anterior cells of the notochord. (BA), embryo before hatching; (BB) and (BC), embryos after hatching showing a complete notochord with 20 cells (blue arrows); (B’) and (B’’)), comparison of pCTor3b-2 activity with the expression pattern of Tor3b-2 env in wild-type embryos. (C) pCTor4b-1 expresses Env in various tissues. The table indicates the number of positive embryos showing expression in the same tissue (Supplementary Figure S7A). (D) Tor4b-1 copies and their envexpression pattern. The table shows the presence of insertions i1 to i14 in F1 animals from individual crosses. Segregation in the progeny was followed using Southern blotting (Supplementary Figure S7B). On the right, micrographs show WISH patterns obtained in the progeny using the antisense Tor4b-1 env probe. Numbers indicate signal frequency.
Mentions: For the synthesis of RNA probes (Supplementary Figure S1), we amplified gene fragments with polymerase chain reaction (PCR) using specific primers and cDNA libraries from mixed developmental stages (1–6 h pf). The PCR products were cloned in pCRII-TOPO (Invitrogen) or pGEM-T vectors (Promega), prior to linearization for in vitro transcription with T7 or SP6 RNA polymerases (Roche). For expression in HEK293T cells, we cloned the complete env ORFs in C-terminal fusions with V5/6His tag in pCDNA 3.1-TOPO vector (Invitrogen). To construct pCTor3b-2 and pCTor4b-1, we generated DNA fragments containing part of env, fused with the V5/6His tag, followed by a stop codon and a restriction site (Figure 6A). These were then digested with the appropriate enzymes and ligated to upstream and downstream DNA fragments in order to reconstruct a Tor element that carried the full gag to the 3′LTR sequence. The resulting inserts were PCR amplified and cloned into the pCRII-TOPO vector.

Bottom Line: Tor Env proteins are membrane-associated glycoproteins which exhibit some features of viral membrane fusion proteins.Such embryonic expression depends on determinants present in the Tor elements and not on their surrounding genomic environment.Our study shows that unusual modes of transcription and expression close to the germline may contribute to the proliferation of Tor elements.

View Article: PubMed Central - PubMed

Affiliation: Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, N-5008, Norway simon.henriet@sars.uib.no.

Show MeSH
Related in: MedlinePlus