Figure 4

Molecular characterization of Nuwa. (a) Illustration shows genomic position, cytological bands, P-element insertion lines, gene span, deficiency lines and Pacman genomic BAC clones related to Nuwa. Reagents tested in this study are shown in orange. (b, bʹ) Genomic DNA isolated from the P¹⁰²⁵²³ insertion line was cut with restriction enzymes (e.g., Sau3A, HhaI and HpaII), self-ligated into circularized DNAs and then used as a template for inverse PCR. The PCR products contained flanking DNA fragments of P¹⁰²⁵²³, which were mapped to three genes, including Fas3 (yellow asterisks; lanes 1, 4 and 7), DIP-θ (green asterisks; lanes 14 and 15) and CG11030 (magenta asterisks; lanes 14 and 15). (c, cʹ) Genomic DNA isolated from P¹⁰²⁵²³, P¹¹¹⁴⁷⁷, FRT^40A and P¹⁰²⁵²³, FRT^40A mutant lines was directly used for PCR reactions to obtain DNA fragments flanking P-element insertions. P-element-flanking DNA fragments mapped to Fas3 (yellow asterisks) in genomic DNA from the P¹⁰²⁵²³ mutant line (lanes 7 and 8), but not in P¹¹¹⁴⁷⁷, FRT^40A (lanes 1 and 2) or P¹⁰²⁵²³, FRT^40A (lanes 4 and 5) mutant lines. In contrast, P-element-flanking DNA fragments mapped to DIP-θ (green asterisks; lanes 3, 6, 9 and 11) and CG11030 (magenta asterisks; lanes 10 and 12) were found in genomic DNA isolated from P¹¹¹⁴⁷⁷, FRT^40A and P¹⁰²⁵²³, FRT^40A mutant lines. Oligos used for reverse PCR and PCR reactions in panels b, bʹ, c, cʹ are listed in Supplemental table 3.