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Blood, Vol. 104, Issue 5, 1498-1503, September 1, 2004
Erythropoietin gene from a teleost fish, Fugu rubripes
Blood Chou et al.
104: 1498
Supplemental materials for: Chou et al, Vol 104, Issue 5, 1498-1503
Files in this Data Supplement:
- Figure S1. Comparison of amino acid sequences of the Fugu and mammalian Epos (PDF file, 60 KB) -
The alignment was generated using the ClustalX program. The total number of residues in each sequence is shown at the end. Residues conserved in all species are indicated by an asterisk (*). The arrowhead indicates the position of introns in Fugu and human genes. The cysteine residues that form disulfide bridges are shown in boldface letters. The N-linked glycosylation sites in the human sequence are marked by circles, and O-linked glycosylation sites in the human and Fugu sequences are boxed. The mammalian Epo sequences were retrieved from GenBank (accession numbers: human, AAF23134; macaque (a) (Rhesus macaque), I84613; macaque (b) (Crab-eating macaque), JQ0173; pig, CAB96417; sheep, I46401; bovine, NP_776334; horse, BAC55239; dog, P33707; cat, P33708; rabbit, JC7699; rat, NP_058697; and mouse, A24902).
- Figure S2. Mapping of transcription start site of Fugu Epo expressed in cell lines (PDF file, 64 KB) -
The PLHC-1 or HepG2 cell line was transfected with an 11-kb fragment of the Fugu cosmid c68C11. Total RNA was extracted from the cells, and single-strand cDNA was synthesized using oligo(dT) primer followed by annealing of the SMARTII oligo to the 5' end (SMART RACE cDNA kit; Clontech). The sequence of the SMARTII oligo serves as a template for the "universal primer." The 5' end of the cDNA was amplified using the universal primer and FEPOR3 primer (5'-AGGCCTCAGGGGGATGGCAG-3'), cloned into a T vector and sequenced. (A) Representative chromatogram of the 5' RACE product. The universal primer sequence is underlined. The transcription start site is indicated by a bent arrow, and intron position is indicated by an arrowhead. The initiation codon is boxed. (B) To ascertain if any transcripts are generated from sites upstream of the transcription site mapped by RACE, we performed RT-PCR using either Primer tss-22 (located 22 bp upstream of the transcription site) or Primer tss+22 (located 22 bp downstream) together with FEPOR3. The absence of a product with tss-22 and the presence of an expected size band (308 bp) with tss+22 in both PLHC-1 and HepG2 cells confirm that no transcripts are generated from upstream sites. (C) Primer tss-22 or tss+22 together with FEPOR3 was used to amplify cosmid c68C11 DNA as a positive control.
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