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Transcriptional and translational regulation of enolase under anaerobic stress in maize
Abstract
A cDNA encoding maize enolase (2-phospho-D-glycerate hydrolase) was isolated by functional genetic complementation using an enolase deficient mutant of Escherichia coli. This cDNA, present on pZM245 contained an open reading frame encoding a protein of 446 amino acids with a high degree of similarity to enolase sequences from other organisms. The specific activity of enolase was found to increase to twice its initial levels after 48 hours of anaerobiosis. Northern blot analysis showed a five fold anaerobic induction of enolase mRNA, while the mRNAs of genes not encoding glycolytic enzymes were reduced to undetectable levels. Southern-blot analysis of maize genomic DNA indicated that there is one copy of the cloned enolase gene per haploid genome of maize. Enolase was purified from maize (Zea mays L. inbred B73) seeds. Purification steps included: ammonium sulfate precipitation, gel filtration, Mono Q and Phenyl Superose chromatography. The purified fraction resolved into 55 and 56 kilodalton proteins by SDS-PAGE. Polyclonal antibodies raised against the fusion protein of pZM245 specifically bound to both purified proteins in all stages of purification. Polyclonal antibodies have also been generated against purified enolase. These antibodies recognize a 56 kilodalton protein in cell extracts of E.coli DF261(pZM245) but not in the cell extracts of E.coli DF261 (enolase deficient mutant of E.coli). These observations suggest that the 55 kilodalton protein is either a modification of the 56 kilodalton polypeptide, or an enolase isozyme. The plastid fraction of maize lacked both enolase protein and activity. Protein levels of enolase remained unchanged under anaerobic stress. In-vivo labeling of the proteins in maize roots revealed that enolase is not translated efficiently under anaerobic stress.
Subject Area
Botany|Genetics|Molecular biology
Recommended Citation
Lal, Shailesh Kumar, "Transcriptional and translational regulation of enolase under anaerobic stress in maize" (1992). ETD collection for University of Nebraska-Lincoln. AAI9314410.
https://digitalcommons.unl.edu/dissertations/AAI9314410