08. A/B/O Allelic cDNAs

 

We initially prepared two additional cDNA libraries using RNAs from human colon carcinoma SW948 and SW48 cell line cells (Yamamoto, 1990). The SW48 cell line was derived from a patient with AB blood type, whereas the SW948 cell line was derived from a different patient with O blood type. We used a probe prepared from the FY59-5 cDNA clone that was isolated from the MKN45 cDNA library and contained a cDNA fragment much larger than 98 bps. We identified clones that were hybridized with the probe, and determined the nucleotide sequences. The cDNA clones from the SW48 cDNA library were divided into 2 groups depending on sequence difference; one group showed more homology to the cDNA sequence of the MKN45 cDNA library than the other group. Therefore, that group was assumed to represent the A allele, whereas the group with more differences was assumed to represent the B allele. Conversely, the cDNA clones from the SW948 cDNA library showed identical nucleotide sequences, though several clones showed different splicing patterns. We realized that, compared with the cDNA clones from the MKN45 cDNA library, the cDNA clones from SW948 had a single nucleotide deletion. Because a single nucleotide in the coding region may change the frame of codons and produce non-functional proteins due to a frameshift, we assumed that those cDNAs represented the O allele.

We then constructed 2 additional cDNA libraries of human colon adenocarcinoma (cell lines COLO205 and SW1417). The COLO205 cell line cells exhibited the O phenotype, though the ABO blood group of the patient was not known. The SW1417 cell line cells were derived from a patient with B blood type and exhibited the B phenotype. The screening of these two cDNA libraries identified dozens of cDNA clones that contained homologous sequences. DNA sequencing was performed, and we determined that the cDNA clones from COLO205 showed an identical nucleotide sequence, though there were variations in the splicing pattern. All of the cDNA clones contained the single nucleotide deletion previously identified in the SW948 cDNA clones. Although the cDNA clones also possessed additional nucleotide substitutions, they were assumed to represent the O allele because of the presence of the single nucleotide deletion. The cDNA clones from SW1417 were divided into 2 groups by differences in the nucleotide sequence. One group showed the same sequence as the cDNAs from SW948, which was assumed to represent the O allele, and other group showed the same sequence as the cDNAs from SW48, which was assumed to represent the B allele. This demonstrated that the blood type B patient from whom the SW1417 cell line cells derived had a BO genotype and not a BB genotype. This experiment became the first successful example of ABO genotyping. Before this discovery, it was impossible to determine the ABO genotype of individuals with blood type A or B by immunological methods.

09. Deduced Amino Acid Sequences of A/B/O Alleles

01. ABO Blood Group System