For the many sequence alignment under.Positions highlighted in dark grey show amino acids identical in a minimum of half of Torb and b sequences, positions highlighted in light grey show conservative substitutions.Fulllength env cDNAs have been obtained for elements whose names are underlined.NeighbourJoining solutions, reproducibly revealed Tor, Tor, Tora, Torb, Tora and Torb groups (Figure A).Their positions relative to other gypsy elements were variable and weakly supported.In Torb components, we systematically found a space among pol and env genes (Figure A).Half in the Tor elements with env belong to Tora, with all the other folks belonging to Torb and b.A few Tor components with out env had been also located in these groups.Biochemical PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21569535 study of Tor Env.The comparison of Env proteins from Torb and b revealed hugely conserved residues, forming motifs absent in Tora.These conserved functions, discovered upstream of a Cterminal transmembrane domain, include a putative fusion tripeptide FXG , a N[LV]G motif flanked by aromatic residues as well as a set of sevencysteines (Figure E and Supplementary Figure S).Employing human HEKT cells, we expressed Torb and b Env proteins fused to a Cterminal tag.Env was connected with cell membranes in the course of subcellular fractionation (Figure B).Immunostaining of transfected cells did not permit conclusive identification from the organelles where these Env proteins had been positioned.We also expressed Torb Env in Oikopleura embryos by microinjection of a reporter construct (see `Tissuespecific expression of Tor is driven by internal regulatory sequences’ of `Results’ section).Within this case, we observed Env localized on the plasma membrane and in perinuclear structures, probably the endoplasmic reticulum (ER) (Figure D).The presence of glycosylated residues on Torb Env expressed in human cells also indicated ER addressing (Figure C).Oligosaccharides in suchNucleic Acids Investigation, , Vol No.was spliced and transcribed in the LTR as well (Supplementary Figure S).We mapped splice donor websites close towards the beginning of gag and splice acceptor internet sites upstream of env.In each situations the intron had a noncanonical donor internet site GA, but an acceptor site similar to these of GTAG introns in the genome .Much more surprisingly, envprimed RACE items from Torb and b elements were systematically devoid of LTR sequences and N-Acetylneuraminic acid Autophagy lacked evidence of splicing (Figure B).In 5 situations, the cDNA matched a sequence beginning upstream of env, either in pol for Torb components or inside the intergenic space that follows pol in Torb elements.Due to the fact our cloning protocol selects for capped RNA, these RACE goods probably represent transcripts synthesized from an internal promoter downstream of the LTR.We refer to this promoter as Penv, and towards the LTR promoter as Pltr.We further characterized env mRNA with northern blotting making use of antisense probes that cover pol, env or the UTR of Torb (Figure C).We identified two transcripts expressed in the embryo.The longer a single corresponds to the gRNA along with the shorter 1 to env mRNA.Additional RACE analyses showed that and ends of Torb gRNA carry RU and UR arrangements, respectively, that are essential for conservative replication .Insertion polymorphism and developmentallyregulated expressionFigure .Characterization of Tor TSSs.(A) Genetic organization of Tor components.LTRs are shown as striped boxes, arrows and numbers indicate Transcription Begin Web sites (TSSs) mapped with RACE.Intron positions and border sequences are shown for Tora components.sd, splice dono.