The transcription of the zein genes presents an interesting regulatory phenomenon since not only are the genes expressed at very high levels but their expression is also developmentally and tissue specific. The use of cDNA clones specific for the major zein protein classes has enabled the quantitation of mRNA levels during development and the identification of precursor forms of the zein mRNAs.
Total RNA isolated from kernels of a growing maize ear was electrophoretically fractionated on agarose gels under strictly denaturing conditions (2.2 M formaldehyde) and transferred to nitrocellulose. Once immobilized on nitrocellulose the zein specific RNAs were identified by hybridization with 32P-labeled zein cDNA probes (Northern technique). The mature zein mRNA was readily seen at its expected size of about 1,000 bases but several other bands were also strongly labeled. These had sizes of about 1,600 bases and greater. The existence of the first of these precursor mRNAs (1,600 bases long) was subsequently confirmed by electron microscopic analysis of RNA:DNA hybrids formed between poly rA+RNA and a genomic clone containing a zein gene. It is an interesting feature of these precursors and possibly indicative of specialized transcriptional control of zein synthesis that the precursor forms of the zein mRNA are present in concentrations approximately equivalent to those of the final sized mRNA.
The Northern technique has also been used to analyze zein mRNA over the developmental period of zein synthesis in both wild-type and opaque-2 mutant endosperms. The opaque-2 mutation reduces the amount of the zein proteins synthesized, particularly the 21,000 dalton proteins. It has now been possible by this method to demonstrate that levels of hybridizable RNA for both major zein protein classes correspond with levels of the proteins in the endosperm. Furthermore, this method has enabled us to exclude processing of the precursor and specific degradation of the zein mRNAs as sites of action of the opaque-2 mutation. It, therefore, appears that the biochemical lesion in opaque-2 is a transcriptional event rather than the previously postulated translational modification. The mechanism and role of the opaque-2 mutation on the transcription of zein genes will require further analysis of the structure and organization of these genes in normal and mutant plants.
P. Langridge, J. Pintor-Toro and G. Feix
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