Diagnosis of retinoblastoma by the use of restriction fragment length polymorphism of esterase D gene

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1. Esterases D-1 and D-2 were highly purified from human erythrocytes and were characterized.2. White rabbits were immunized with the purified esterase D-1. The antibodies to esterase D-1 reacted specifically to esterases D.3. The purified esterase D-1 was digested with proteolytic enzymes. The resulting peptides were separated by high performance liquid chromatography. The primary structures of nine purified peptides were analyzed.4. cDNA library was prepared from human placenta and was screened with the antibody against esterase D-1. one positive clone was isolated.5. The insert corresponding to cDNA, approximatelly 300 base paris, was subcloned into pUC 9.6. The base sequence of the cDNA was determined by the dideoxy method. It encodes the amino acid sequence determined in 3.7. The original cDNA library prepared from human placenta was rescreened with the cDNA as a probe. The cDNA clone with 1,200 base pairs as an insert was obtained.8. The base sequence of the longer cDNA showed no methionine codon at the 5'-terminal portion, suggesting that the longer cDNA lacks the 5'-terminal portion encoding approximate 10 amino acids at the amino terminal.