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  • 1
    ISSN: 1573-5028
    Keywords: Coix ; coixin genes ; 22kDa-like α-prolamin ; storage protein ; opaque-2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Several genomic and cDNA clones encoding the 22 kDa-like α-coixin, the α-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like α-coixin genes designated α-3A, α-3B and α-3C were found in the 15 kb α-3 genomic clone. The α-3A and α-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the α-3B gene, suggesting that the three α-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 α-coixin clones with the published sequences of 22 kDa α-zein and 22 kDa-like α-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 α-prolamins and the 19 kDa α-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 α-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. −300 prolamin box are present at conserved positions in α-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in α-3B that occupies approximately the same positions as those identified for the 22 kDa α-zein and α-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 α-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.
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  • 2
    ISSN: 1573-5028
    Keywords: maize ; Coix ; zein ; coixin ; phylogenetic relationship
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain α-, β-, and γ-zein and α-, β-, and γ-coixin. The α-coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa α-zeins. Like the α-zeins, the C1 and C2 α-coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to γ-coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of γ-zein and represents 15% of the total coixin. The β-zein fraction was composed of a major 17 kDa protein band, while the β-coixin fraction consisted of a mixture of α- and γ-coixins. Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa α-zein, as did C4 and C5 antisera. The antiserum against γ-coixin showed strong cross-reaction with γ-zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa α-zeins as well as the 28 and 16 kDa γ-zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa α-zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa γ-zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa γ-zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.
    Type of Medium: Electronic Resource
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