We have isolated several α-amylase genomic clones from an Eco RI library of barley DNA in λ-Charon 32. Five of these clones exhibit unique restriction maps and differences in their abilities to hybridize with two previously characterized α-amylase cDNA probes representing two different loci, α-Amy 1 (high pI) and α-Amy 2 (low pI) on barley chromosomes 6 and 1, respectively. Stringent hybridizations indicate that four of the five genomic clones contain α-Amy 1 sequences and one contains α-Amy 2 sequences. The regions containing α-amylase genes from one representative genomic clone of each group have been sub-cloned, mapped and sequenced. S1-nuclease protection experiments indicate that the two α-amylase genes contained in these clones are functional in aleurone tissue. Transcription start sites in these genes were determined by primer extension using specific synthetic oligonucleotide primers.
The DNA sequences of the two α-amylase genes, including promoter regions, are divergent, as are the predicted amino acid sequences of the mature proteins and the N-terminal “leader” peptides. The α-Amy 1 gene contains two introns while the α-Amy 2 gene has three introns. In the coding region, each gene shows 7–10% sequence divergence with respect to the previously characterized cDNA clones of the same gene type. Therefore, differences in nucleotide sequences can account for some of the isozyme variations seen between the sub-families of α-amylases and among members of the same subfamily. Although the nucleotide sequences of the promoter regions of α-Amy 1 and α-Amy 2 genes show little homology, both contain pairs of inverted repeat elements which could constitute regulatory sites.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Benton WD, Davis RW: Screening λ-gt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182 (1977).
Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513–1523 (1979).
Brown AHD, Jacobsen JV: Genetic basis and natural variation of α-amylase isozymes in barley. Genet Res (Camb) 40: 315–324 (1982).
Callis J, Ho T-HD: Multiple molecular forms of the gibberellin-induced α-amylase from the aleurone layers of barley seeds. Arch Biochem Biophys 224: 224–234 (1983).
Chandler PM, Zwar JA, Jacobsen JV, Higgins TJV, Inglis AS: The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers-studies using an α-amylase cDNa clone. Plant Molec Biol 3: 407–418 (1984).
Deikman J, Jones RL: Control of α-amylase mRNA accumulation by gibberellic acid and calcium in barley aleurone layers. Plant Physiol 78: 192–198 (1985).
Dynan WS, Tjian R: Control of eukaryotic messenger RNA synthetis by sequence-specific DNA-binding proteins. Nature 316: 774–778 (1985).
Favaloro J, Treisman R, Kamen R: Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol 65: 718–749 (1980).
Higgins TJV, Jacobsen JV, Zwar JA: Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers. Plant Molec Biol 1: 191–215 (1982).
Huang J-K, Swegle M, Dandekar AM, Muthukrishnan S: Expression and regulation of α-amylase gene family in barley aleurones. Exp J Mol Appl Genet 2: 579–588 (1984).
Jacobsen JV, Higgins TJV: Characterization of the α-amylases synthesized by aleurone layers of Himalaya barley in response to gibberellic acid. Plant Physiol 70: 1647–1653 (1982).
Loenen WAM, Blattner FR: Lambda Charon vectors (Ch 32, 33, 34 and 35) adapted for DNA cloning in recombination deficient hosts. Gene 26: 171–179 (1983).
McKnight SL, Kingsbury RC, Spence A, Smith M: The distal transcription signals of the herpesvirus tk gene share a common hexanucleotide control sequence. Cell 37: 253–262 (1984).
Messing J: New M13 vectors for cloning. Methods Enzymol 101: 20–78 (1983).
Mount SM: A catalogue of splice junction sequences. Nucleic Acids Res 10: 459–472 (1982).
Muthukrishnan S, Chandra GR, Albaugh GP: Modulation by abscisic acid and S-2-aminoethyl-L-cysteine of α-amylase mRNA in barley aleurone cells. Plant Molec Biol 2: 249–258 (1983).
Muthukrishnan S, Chandra GR, Maxwell ES: Hormone-induced increase in levels of functional mRNA and α-amylase mRNA in barley aleurones. Proc Natl Acad Sci USA 76: 6181–6185 (1979).
Muthukrishnan S, Chandra GR, Maxwell ES: Hormonal control of α-amylase gene expression in barley studies using a cloned cDNA probe. J Biol Chem 258: 2370–2375 (1983).
Muthukrishnan S, Gill BS, Swegle M, Chandra GR: Structural genes for α-amylases are located on barley chromosomes 1 and 6. J Biol Chem 259: 13637–13639 (1983).
Norrander J, Kempe T, Messing J: Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26: 101–106 (1983).
Rogers JC: Two barley α-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260: 3731–3738 (1985).
Rogers JC, Milliman C: Isolation and sequence analysis of a barley α-amylase cDNA clone. J Biol Chem 258: 8169–8174 (1983).
Rogers JC, Milliman C: Coordinate increase in major transcripts from the high pI α-amylase multigene family in barley aleurone cells stimulated with gibberellic acid. J Biol Chem 259: 12234–12240 (1984).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Svensson B, Mundy J, Gibson RM, Svendsen I: Partial amino acid sequences of α-amylase isozymes from barley malt. Carlsberg Res Commun 50: 15–22 (1985).
Varner JE, Chandra GR: Hormonal control of enzyme synthesis in barley endosperm. Proc Natl Acad Sci USA 52: 100–106 (1964).
Wilbur WJ, Lipman DJ: Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci USA 80: 726–730 (1983).
About this article
Cite this article
Knox, C.A.P., Sonthayanon, B., Chandra, G.R. et al. Structure and organization of two divergent α-amylase genes from barley. Plant Mol Biol 9, 3–17 (1987). https://doi.org/10.1007/BF00017982
- α-amylase genes
- DNA sequence
- genomic clones
- transcription starts