|Budget Amount *help
¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1997 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1996 : ¥700,000 (Direct Cost : ¥700,000)
The structural unit of Indoplanorbis hemoglobin is a multidomain polypeptide chain of 10 heme binding domains linked in tandem. 1. The native hemoglobin was cleaved with subtilisin and gel filtrated on Sephacryl S-200. Four heme-containing fractions were obtained at protein/protease ratio of 200 and 50, and MW of the fractions were 22.3kDa(I), 50.6kDa(II), 122kDa(III) and 300kDa(IV). These are tentatively designated as mono-, di-, tetra-, and octa- domain units, respectively. The monodomain fraction showed a heterogeneous pattern by PAGE, containing at least 13 components, and several of them were isolated by Mono Q chromatography. One of the isolated components was sequenced, and determined 20 N-terminal amino acid residues. 2. mRNA was extracted from the whole tissue of Indoplanorbis, and constructed the cDNA library. 3. On the basis of the amino acid sequence 1-11 and 11-17 from the N-terminus, two oligonucleotide probes Ipglob-1 and Ipglob-2 were synthesized, and the cDNA library was screened by a combination of either of the probes with vector primer. The resultant PCR products of several different sizes were sequenced, however, their base sequences were not that of the hemoglobin. 4. On the assumption that this hemoglobin is the repeated sequence of the same or a similar domain, the antisense probes Ipglob-1a for Ipglob-1 and Ipglob-2a for Ipglob-2 were synthesized, and the screening of the hemoglobin cDNA was tried by the primer combination of 1a with 2, or the combination of 1 with 2a. However, this procedure could not obtain any PCR products. 5. Rabbit anti-hemoglobin antibody screening also could obtain no specific signals. 6. In the future, another probe synthesized from the information of the N-terminal amino acid sequence of the 10 domain polypeptide chain will be prepared and the screening will be tried in the same manner as that of 3 and 4.