The discovery of immunostimulatory DNA sequence
Since the 1960s, Mycobacterium bovis BCG has been investigated widely within the scope of cancer immunotherapy of experimental animals and humans [10, 19, 20]. Efforts have also been made to isolate the bacterial component possessing antitumor activity from BCG. While trying to obtain water-soluble components of BCG, we found that BCG cytoplasm precipitated by streptomycin sulfate contained substances strongly active against Line-10 hepatoma of Strain-2 guinea pigs [17, 21]. The streptomycin sulfate precipitate of BCG was a complex of various components, protein, nucleic acid, lipid and sugar. Repeated injections of this fraction into guinea pigs caused severe anaphylactic shock. In one approach, we tried to extract fractions of streptomycin sulfate precipitate with hot water and found that the heat extract contained substances that strongly inhibited tumor growth. This fraction was further purified with multi-step procedures, and a final fraction designated MY-1 was obtained. The chemical composition of MY-1 and the other fractions obtained in the process of isolating MY-1, as well as that of an RNase digest of MY-1 and a DNase digest of MY-1 was examined. MY-1 was composed of 98% nucleic acid (70% DNA, 28.0% RNA) and only 1.3% protein. The sugar content of MY-1 was 0.2% when measured by gas-liquid chromatography. The RNase digest of MY-1 contained mostly DNA (97.0%), and the DNase digest of MY-1 was composed of RNA (98.5%). MY-1 did not contain any unusual amino acids. The diaminopimeric acid content was less than 0.01% and no hexosamines were detected. Of the neutral sugars, only glucose was detected at 0.20%. These results suggested that contamination of MY-1 with cell wall components or Polysaccharides was negligible. The base composition of the RNase digest of MY-1 and of the DNA extracted and purified from BCG by the method of Marmur were examined. The GC contents were 69.8% and 71.5%, respectively, and their base compositions were similar. The DNA contained in MY-1 was single stranded as judged by the results of an ultracentrifuge analysis, chromatography on a hydroxy apatite column, and measurement of temperature absorbance.
KeywordsNatural Killer Activity Mycobacterium Bovis Palindromic Sequence Immunostimulatory Activity Mouse Spleen Cell
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