This inversion event explains the inverse orientation of the CτA-4 sequence in IGH-A.
As previously mentioned [50], the human ALP/Enigma genes cluster specifically in a way that an ALP-like gene pairs with an Enigma-like gene in inverse orientation, analogous to the even-skipped genes, which both (EVX1 and EVX2) are transcribed in an opposite orientation as compared to adjacent HOX genes [52].
However in fish genomes, this locus contains two additional conserved markers ANKRD5 and EMILIN1 (Table S4) flanking in inverse orientation.
Although the inverse orientation has been assumed to be typical of all insect ORs, this remains to be formally demonstrated.
When these recognition sites are placed to the far 5' and 3' end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly.
In fish, the marker sets were not totally conserved: on one side, the CITED4 gene was present, on the other side, two additional flanking genes MANEAL and SF3A3 (Table S4) were present in inverse orientation to the conserved LIN28.
The pol sequence also contains multiple copies of various 30 500 bp repeats; applying a 90% sequence similarity criterion to define a repeat, these sequences account for 4.75% of the genome and are arranged in both direct and inverse orientation.
The FluB minigenome expression plasmid, pPolI-lucRT_B, was obtained by cloning the firefly luciferase ORF (inverse orientation) flanked by the non-coding region of the segment 8 of the B/Yamagata/73 into the SapI-digested plasmid pPolI-SapI-Rib [50].
To additionally avoid the degradation of the BAC in the following transposition procedure, the pBeloBAC11 vector elements were engineered in inverse orientation between the viral sequences with respect to the present mini-F fragment of pHJOFep (Fig. 1B and 1C).
A similar scheme was used to construct Inv2 which employed the same 19-nt sequence, but in the inverse orientation (Figure 1A), a type of scrambled sequence that was routinely used as a control for RNAi experiments.
