Cloning and Complete Nucleotide Sequence of a Full-Length cDNA Encoding a Catalytically Functional Tumor-Associated Aldehyde Dehydrogenase

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 b...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1988-03, Vol.85 (6), p.1782-1786
Main Authors: Jones, David E., Brennan, Mark D., Hempel, John, Lindahl, Ronald
Format: Article
Language:English
Subjects:
Aldehyde Dehydrogenase - genetics
Aldehydes
Amino Acid Sequence
Amino acids
Animals
Base Sequence
Biological and medical sciences
Biotechnology
Cloning, Molecular
Complementary DNA
Deoxyribonucleases, Type II Site-Specific
DNA - analysis
DNA Restriction Enzymes - metabolism
Enzymes
Fundamental and applied biological sciences. Psychology
Gels
Genetic engineering
Genetic technics
Liver
Liver Neoplasms, Experimental - enzymology
Methods. Procedures. Technologies
Molecular Sequence Data
Nucleotides
Phenotype
Rats
RNA
Tumors
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Summary: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 >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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.85.6.1782