Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Abstract

Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

“…these three plant species is reflected in the extensive sequence conservation, in both coding and regulatory regions, that exists among the 22-kD o~-zein genes of maize, an cx-kafirin gene from sorghum, and an cx-coixin gene from Coix (Ottoboni et al, 1993). In view of the conservation of putative cis elements in the promoters of these 22-kD-like o~-prolamin genes, the presence of O2-related proteins in Coix and sorghum is not surprising.…”

N-terminal amino acid sequences of prolamins encoded by the alleles at the Pro1 and Pro2 loci in foxtail millet, Setaria italica (L.) P. Beauv.

“…b Reference: data of prolamin of Echinochloa crus-galli from Chen et al (1997), 22kDa α-zein, α-kafirin and α-coixin from Ottoboni et al (1993), 19kDa α-zein from Marks et al (1985) and pennisetin (prolamin of peral millet) from Bietz (1982). c Valine and leucine were detected at residue 8 with molar ratio of 1.75/1, and isoleucine and valine were found at position 23 with molar ratio of 2.75/1.…”

“…This suggests that alleles at the Pro1 locus and those at the Pro2 locus have not arisen from an identical ancestral gene. The x-type and y-type prolamins encoded by alleles at the Pro2 locus showed homology with prolamin of Echinochloa crus-galli (83.3% with the x-type prolamin and 72.7% with the y-type prolamin, respectively) (Chen et al, 1997) and α -zein-like prolamins of maize (zein) (for 22kDa, 57.1% and 61.5%; for 19kDa, 50.0% and 46.2%), sorghum (kafirin) (57.1% and 53.8%) and Job's tears (coixin) (64.3% and 46.2%), assuming five to six residues as gaps (Ottoboni et al, 1993;Marks et al, 1985). Therefore, the x-type and y-type prolamins were designated as "α-zein-like prolamin".…”

Cooperative DNA Binding and Sequence Discrimination by the Opaque2 bZIP Factor

“…One of these mutants, opaque2 ( o2 ), affects a DNA binding protein belonging to the basic leucine zipper (bZIP) class of transcriptional activators (Hartings et al, 1989;Schmidt et al, 1990). Coix and sorghum contain homologous O2 genes (Ottoboni et al, 1993;Pirovano et al, 1994;Vettore et al, 1998), indicating that this regulatory locus is conserved among these species.…”

“…One of these mutants, opaque2 ( o2 ), affects a DNA binding protein belonging to the basic leucine zipper (bZIP) class of transcriptional activators (Hartings et al, 1989;Schmidt et al, 1990). Coix and sorghum contain homologous O2 genes (Ottoboni et al, 1993;Pirovano et al, 1994;Vettore et al, 1998), indicating that this regulatory locus is conserved among these species.O2 has been shown to activate the transcription of several maize and Coix endosperm genes through binding to significantly different cis -acting elements located in their promoters (Lohmer et al, 1991;Schmidt et al, 1992;Yunes et al, 1994b; Cord Neto et al, 1995;Maddaloni et al, 1996;Muth et al, 1996). Functional O2 binding sites have also been found in the seed-specific genes of pea lectin (de Pater et al., 1994), wheat prolamin (Holdsworth et al, 1995), and French bean phytohemagglutinin (Chern et al, 1996).…”