Fig. 3. Transient expression of Chaenocephalus aceratus and Chionodraco rastrospinosus promoter constructs in C. aceratus pectoral abductor muscle. (A) Schematic depiction of the upstream C. rastrospinosus myoglobin (Mb) promoter constructs (pR1L, pR2L and pR3L) and C. aceratus Mb promoter constructs (pA1L, pA2L and pA3L) generated as described in Materials and methods for use in the in vivo transient expression assay shown in (B). The upstream Mb promoter sequences inserted into the pGL2 basic luciferase plasmid are indicated numerically and are shown in Small et al. (1998). The position of the conserved TATAAAA element is indicated by a triangle at position -25 in all constructs. The putative E-box element found in the upstream promoter sequence of both species and the TATAAAA duplication found in C. aceratus are also indicated in constructs pR1L and pA1L. Promoter constructs were prepared as described in Materials and methods. (B) 1 µg of each test plasmid and 0.5 µg of the Renilla luciferase internal reference construct driven by the cytomegalovirus (CMV) promoter was injected into the pectoral adductor muscle of a C. aceratus individual. Activity of firefly luciferase in each tissue sample was normalized to that of Renilla luciferase. Values are means ± S.E.M. for N=7 trials. Expression of `full-length' pR1L was significantly greater (P<0.001) than all other channichthyid Mb promoter constructs and was not significantly different from the positive control driven by the pSV40Luc promoter. Expression of `full-length' pA1L was not significantly different from any truncated constructs from either species (pR2L, pR3L, pA2L or pA3L). `No reporter' shows expression of the pGL2 luciferase plasmid lacking a promoter sequence.