Abstract
The homodimeric alcohol dehydrogenase gene product of maize (Zea mays L.)Adh1-1S1108 mutation was purified and compared with the parentalAdh1-1S enzyme. The mutant alcohol dehydrogenase activity had pH optima and substrate specificity similar to those of the parental enzyme, but exhibited somewhat increased and decreasedK mvalues for acetaldehyde and NADH, respectively. The mutant enzyme was also markedly less stable than the enzyme from parental tissues to temperatures as low as 50°C. Sequence analysis of a polymerase chain reaction (PCR)-generated cDNA clone revealed a G-to-C mutation at position 406 and a C-to-T mutation at position 974. These would result in residue 103 of each protein subunit being changed from an alanine to a proline and residue 292 being changed from an alanine to a valine. Whether one or both of these changes in primary sequence is responsible for the altered substrate affinities and stability is not yet understood.
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References
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein-dye binding.Anal. Biochem. 72248.
Branden, C.-I., Eklund, H., Cambillau, C., and Pryor, A. J. (1984). Correlation of exons with structural domains in alcohol dehydrogenase.EMBO J. 31307.
Brumbaugh, J. A., Middendorf, L. R., Grone, D. L., and Ruth, J. L. (1988). Continuous, on-line DNA sequencing using oligonucleotide prumers with multiple fluorophores.Proc. Natl. Acad. Sci. USA 855610.
Coulondre, C., and Miller, J. H. (1977). Genetic studies of the lac repressor. IV. Mutagenenic specificity in thelacI gene ofE. coli. J. Mol. Biol. 117577.
Dennis, E. S., Gerlach, W. L., Pryor, A. J., Bennetzen, J. L., Inglis, A., Llewellyn, D., Sachs, M. M., Ferl, R. J., and Peacock, W. J. (1984). Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize.Nucleic Acid Res. 123983.
Devereux, J., Haebeshi, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX.Nucleic Acids Res. 12387.
Eklund, H., Nordstrom, B., Zeppezauer, E., Soderlund, G., Ohlsson, I., Boiwe, T., Soderberg, B.-O., Tapia, O., Branden, C.-I., and Akeson, A. (1976). Three-dimensional structure of horse liver alcohol dehydrogenase at 2.4 Å resolution.J. Mol. Biol. 10227.
Freeling, M., and Bennett, C. D. (1985). MaizeAdh1.Annu. Rev. Genet. 19297.
Good, A., Pelcher, L. E., and Crosby, W. L. (1988). Nucleotide sequence of a complete barley ADH1 cDNA.Nucleic Acids Res. 167182.
Ha, D. B. D., Buffard, D., Berger, F., Breda, C., and Esnault, R. (1990). Nucleotide sequence encoding a slow allele ofAdh1 in pearl millet.Plant Mol. Biol. 14453.
Irish, E. E., and Schwartz, D. (1987). Activation of low and null activity isozymes of maize alcohol dehydrogenase by antibodies.Mol. Gen. Genet. 208271.
Kelly, J., and Freeling, M. (1980). Purification of maize alcohol dehydrogenase-1 allozymes and comparison of their tryptic peptides.Biochim. Biophys. Acta 624102.
Lebkowski, J. S., Miller, J. H., and Calos, M. P. (1986). Determination of DNA sequence changes induced by ethyl methanesulfonate in human cells, using a shuttle vector system.Mol. Cell. Biol. 61838–1842.
MacDonald, R. J., Swift, G. H., Przybyla, A. E., and Chirgwin, J. M. (1987). Isolation of RNA using guanidinium salts. In Berger, S. L., and Kimmel, A. R. (eds.),Guide to Molecular Cloning Techniques Academic Press, San Diego, pp. 219–227.
Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991). Construction of T-vectors, a rapid and general system for direct cloing of unmodified PCR products.Nucleic Acids Res. 191154.
Osterman, J. C., and Dennis, E. S. (1989). Molecular analysis of theAdh1-Cm allele of maize.Plant Mol. Biol. 13203.
Sachs, M. M., Dennis, E. S., Gerlach, W. L., and Peacock, W. J. (1986). Two alleles of maize alcohol dehydrogenase 1 have 3′ structural and poly(A) additiona polymorphisms.Genetics 113449.
Schwartz, D. (1971). Subunit interactions of a temperature-sensitive alcohol dehydrogenase mutant in maize.Genetics 67515.
Schwartz, D. (1975). The molecular basis for allelic complementation of alcohol dehydrogenase mutants of maize.Genetics 79207.
Xie, Y., and Wu, R. (1989). Rice alcohol dehydrogenase genes: Anaerobic induction, organ specific expression and characterization of cDNA clones.Plant Mol. Biol. 1353.
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Funding for this research was from a University of Nebraska—Lincoln Research Council Grant-in-Aid and from the UNL Center for Biotechnology. Sequencing was carried out by the UNL Center for Biotechnology Core Facility for DNA Sequencing. Computing costs were supported by the UNL Computer Resource Center.
The sequence forAdh1-1S1108 is available as Gen-Bank accession L23548.
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Osterman, J.C., Chiang, Y. & Markwell, J. Characterization of mutation-induced changes in the maize (Zea mays L.) ADH1-1S1108 alcohol dehydrogenase. Biochem Genet 31, 497–506 (1993). https://doi.org/10.1007/BF02426881
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DOI: https://doi.org/10.1007/BF02426881