Coordinate increase in major transcripts from the high pI alpha-amylase multigene family in barley aleurone cells stimulated with gibberellic acid.

Abstract

The purpose of this study was to identify specifically genes and transcripts for the high pI isozyme of barley alpha-amylase. From hybridization of coding sequence probes to blots of genomic DNA digested with restriction enzymes that do not cut within our cloned high pI alpha-amylase cDNA, it is estimated that about 7 alpha-amylase genes or pseudogenes exist. No difference could be detected between barley aleurone cell and sprout DNAs. Experiments using probes from the 5' and 3' untranslated sequences of the high pI alpha-amylase cDNA clone identified three HindIII fragments that probably carry high pI sequences. Primer extension experiments used as a primer the terminal 5' coding sequence from our cDNA clone; this primer would not cross-hybridize to low pI alpha-amylase transcripts. Two major transcripts were identified. These shared a conserved 23-base sequence immediately 5' to the ATG start codon, although a C----G transversion and a 3-base deletion were present within this sequence. An unusual 8-base pair GC palindrome was present in the conserved region immediately preceding the ATG start codon. Distal to the conserved sequence there was no apparent homology. One transcript carrying a 97-base untranslated region was identical to our high pI cDNA clone E. The gene for the other was recovered from a lambda phage genomic library. The 5' coding sequence was very similar, but not identical to clone E, demonstrating that these transcripts arise from separate genes. The two transcripts increased coordinately in aleurone cells stimulated with gibberellic acid. These data indicate that there is a high pI alpha-amylase multigene family with at least two active members, both of which are regulated in some manner by the plant hormone gibberellic acid.