Barley alpha-amylase genes. Quantitative comparison of steady-state mRNA levels from individual members of the two different families expressed in aleurone cells

We have cloned and sequenced two barley alpha-amylase genes belonging to the high pI isozyme family, one of which, Amy6-4, corresponds to a cDNA previously characterized by our laboratory. A 750-base pair probe from Amy6-4, representing primarily the promoter/upstream sequences cross-hybridizes on genomic Southern blots under stringent conditions to five other genes or pseudogenes; this demonstrates that the promoter/upstream region in these different members of the gene family is highly conserved. In contrast, this probe hybridizes very poorly to the genomic fragment containing the other cloned high pI gene, Amy46, a finding consistent with substantial divergence of sequence about 200 base pairs upstream from the TATA box of each. We compared steady-state mRNA levels from these individual genes to levels for mRNAs from two low pI alpha-amylase genes and from the single copy gene for aleurain, a thiol protease, using quantitative S1 nuclease protection assays. We found, in RNA from aleurone cells treated with gibberellic acid for 19-24 h, that the two low pI alpha-amylase mRNAs are each about five times more abundant than Amy6-4 or aleurain, which are, in turn, about 10 times more abundant than Amy46. These results indicate that as many as seven closely related high pI genes are needed to provide mRNA levels approaching those from the two low pI genes. We speculate that the substantially lower level of expression of Amy46 may be related to its divergent sequence upstream from the promoter.