Figure 2. Complete nucleotide sequence for the HbIII cDNA.

The overlapping sequences of degenerate RT-PCR products with the 5′ RACE and 3′ RACE products were used to obtain the complete nucleotide sequence for the HbIII cDNA. Most of the HbIII cDNA sequence (97%) was confirmed by end to end RT-PCR. A. Schematic Organization of cDNA Fragments. An initial 342-nt cDNA clone encoding 114 amino acid residues of HbIII (amino acid 26 to 139) was amplified from total RNA using degenerate oligonucleotides. The 5′ RACE product contained a cDNA sequence encoding 43 amino acid residues of HbIII (amino acids 1 to 43) and 62 nt of the 5′ UTR. The 3′ RACE products contained a cDNA sequence encoding 71 amino acid residues of HbIII (amino acid 82 to 152) and two 3′UTR regions (168 and 355 nt long). The numbers at each end of the boxes identify the amino acid numbers of the HbIII aminoacid sequence included in the amplification product. Numbers in parentheses indicates the length in nucleotides of the cDNA regions indicated. Black boxes represent the coding regions and white boxes the UTRs. B. Schematic diagram of the HbIII cDNA. The untranslated region (5′UTR) is composed of 62 nt whereas two 3′UTR regions were isolated revealing the presence of alternative polyadenylation site. The shortest 3′UTR is composed of 168nt out of 689 nucleotides-long cDNA. The longest 3′UTR (355 nt long) is found in 876 nt-long cDNA. The PA indicates the poly-adenylation signal. Numbers in parentheses indicate the length in nucleotides of the cDNA regions indicated. C. Full length cDNA Sequence and Derived Amino Acid Sequence of Hemoglobin III from Lucina pectinata. A single amino acid difference (Asn72Asp) with the reported Lucina pectinata HbIII amino sequence is shown in bold. The two polyadenylation signals are underlined. The ▽ symbol represents the site where the poly (A)+ tail is added in the smaller cDNA. Primers used in RT-PCR, RACE and Primer Extension experiments are shown.