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The myoglobin gene of the Antarctic icefish, Chaenocephalus aceratus, contains a duplicated TATAAAA sequence that interferes with transcription

Deena J. Small1,2,*, Thomas Moylan1,{dagger}, Michael E. Vayda1,2 and Bruce D. Sidell1,2,{ddagger}

1 School of Marine Sciences, University of Maine, Orono, ME 04469, USA
2 Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469, USA
* Present address: Maine Medical Center Research Institute, Center for Molecular Medicine, 81 Research Drive, Scarborough, ME 04074, USA
{dagger} Present address: Marine Education and Research Center, c/o Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA 93407, USA



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  		Fig. 1. Binding of HeLa cell transcription factor TFIID to icefish myoglobin (Mb) promoter sequences. (A) Oligonucleotides corresponding to both strands of the Chaenocephalus aceratus upstream promoter sequence (-715 to -674) that contains the TATAAA duplication (in bold) (C. aceratus upstream promoter oligo), the homologous region (-666 to -640) of the C. rastrospinosus promoter (C. rastrospinosus upstream promoter oligo) and the conserved promoter sequence (-45 to -4) containing the putative `TATAAAA element' located within the core promoter region of both icefish (conserved TATAAAA element oligo) were synthesized based on the upstream promoter sequences obtained as described in Small et al., 1998Go. Numeric positions of sequences are relative to the transcription start site of each species. The TATAAAA sequence for both the C. aceratus upstream oligo and the conserved TATAAAA element oligo are underlined. Prior to use in the gel mobility shift assays, the complementary oligonucleotides were annealed and 32P-end labeled using T4 polynucleotide kinase and [{gamma}-32P]ATP prepared as described in the Materials and methods. (B) 2 pmol of the radiolabeled conserved TATAAAA element oligo (32P-TATAAAA, lanes 1-5), radiolabeled C. aceratus upstream promoter oligo (32P-Ace, lanes 6-10) or radiolabeled C. rastrospinosus upstream oligo (32P-Ras, lanes 11 and 12) were incubated with (+) or without (-) 20 ng of purified human TFIID as indicated. Some samples also included 200 pmol of unlabeled conserved TATAAAA oligo (TATAAAA, lanes 3 and 7), C. aceratus upstream promoter oligo (Ace, lanes 4 and 8) or C. rastrospinosus upstream oligo (Ras, lanes 5 and 9) as competitor oligonucleotides. The DNA/TFIID complexes were then resolved by electrophoresis and autoradiography as described under Materials and methods. Arrows indicate the positions of shifted bands that presumably represent radiolabeled DNA/TFIID complexes.

 


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  		Fig. 2. Binding of factors present in Chionodraco rastrospinosus cardiac muscle nuclear extracts to myoglobin (Mb) promoter sequences. (A) 2 pmol of labeled Chaenocephalus aceratus upstream promoter sequence (-715 to -674) containing the TATAAA duplication shown in Fig. 1A were incubated with either 2 ng HeLa transcription factor IID (TFIID) (lane 1) or 5 µg of a C. rastrospinosus cardiac muscle nuclear extract (lanes 3-6) prepared as described under Materials and methods. No retardation bands were evident in the absence of added protein (lane 2). 200 pmol of unlabeled conserved TATAAAA oligo (TATAAAA, lane 5), C. aceratus upstream promoter oligo (Ace, lane 4) or C. rastrospinosus upstream oligo (Ras, lane 6) were used as competitor oligonucleotides as indicated. DNA/nuclear protein complexes were then resolved by electrophoresis and autoradiography as described under Materials and methods. (B) 2 pmol of labeled conserved TATAAAA element oligonucleotides (32P-TATAAAA) shown in Fig. 1A were incubated with 5 µg of a C. rastrospinosus cardiac muscle nuclear extract prepared as described under Materials and methods. Retardation bands generated by incubation of the 32P-labeled conserved TATAAAA sequence with nuclear extracts from C. aceratus are shown in lane 1. 200 pmol of unlabeled conserved TATAAAA oligo (TATAAAA, lane 2), C. aceratus upstream promoter oligonucleotide (Ace, lane 3) or C. rastrospinosus upstream oligonucleotide (Ras, lane 4) were used as competitor oligonucleotides as indicated. DNA/nuclear protein complexes were then resolved by electrophoresis and autoradiography as described under Materials and methods. Arrows denote DNA/protein complexes that are eliminated by 200 pmol of unlabeled C. aceratus upstream promoter oligonucleotide.

 


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  		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. (1998Go). 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.

 

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