To study the mechanism(s) controlling expression of the
tumor-associated
aldehyde dehydrogenase (
tumor ALDH), which appears during rat hepatocarcinogenesis, cDNAs encoding this
isozyme were cloned and identified with an antibody probe.
Poly(A)-containing
RNA from HTC rat
hepatoma cells, which have been shown to possess high levels of
tumor ALDH, was used as template to synthesize double-stranded
cDNA. The
cDNA was methylated to protect internal sites. Two different synthetic
DNA linkers were added sequentially to the
cDNA to insure correct orientation for expression from the lac promoter of pUC8. A library of 100,000 independent members carrying inserts greater than 1 kilobase was obtained. From this library, two apparently identical
tumor ALDH clones, differing only in size, were identified with an indirect immunological probe. The larger of the
cDNA clones identified, pTALDH, was chosen for further study. Interestingly, since
tumor ALDH is a dimeric
enzyme, pTALDH directs synthesis of a functional
tumor ALDH in the bacterial cell. The
cDNA sequence has been confirmed by comparison to the amino acid sequence of
tumor ALDH purified from HTC cells.