06. A transferase cDNA cloning


In 1990, we cloned cDNAs encoding human A transferase (Yamamoto et al., 1990) based on the partial amino acid sequence of the isolated protein (Clausen et al., 1990). We reverse-translated the amino acid sequences of the tryptic peptides from the isolated protein into nucleotide sequences. Based on the sequence information, we prepared 3 degenerate oligonucleotides, 2 of which were later used as PCR primers. The remaining oligonucleotide for the internal sequence was used as a probe. After RT-PCR of RNA prepared from human stomach cancer MKN45 cell line cells, which are known to express large amounts of A antigens and a high activity of A transferase, the PCR products were electrophoresed through an agarose gel. The DNA was denatured, neutralized, and Southern transferred onto a nitrocellulose membrane. It was then fixed and subsequently hybridized with the the 32P-radiolabelled oligonucleotide probe for the internal sequence. Although the PCR products exhibited smears of bands in the agarose gel, a single band of the DNA fragment of the expected band size of 98 base pairs (bps) was hybridized. As such, the band was cut, PCR-amplified, and cloned into a plasmid vector. After transformation of E. coli, bacterial clones that contained the plasmid with a correct insert were identified. The 98 bp DNA fragment was then used as a probe to screen the MKN45 cDNA library prepared in the lambda 10 phage vector.

07. Northern Hybridization Results

01. ABO Blood Group System

Clausen, H., White, T., Takio, K., Titani, K., Stroud, M., Holmes, E., Karkov, J., Thim, L., and Hakomori, S. (1990). Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1->2Gal alpha 1->3-N-acetylgalactosaminyltransferase from human lung tissue. J Biol Chem 265, 1139-1145. (http://www.jbc.org/cgi/reprint/265/2/1139)

Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S. (1990). Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1-2Gal alpha 1-3GalNAc transferase (histo-blood group A transferase) mRNA. J Biol Chem 265, 1146-1151. (http://www.jbc.org/cgi/reprint/265/2/1146)


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