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GrainGenes Reference Report: TAG-104-828

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Reference	TAG-104-828
Title	Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica
Journal	Theoretical and Applied Genetics
Year	2002
Volume	104
Pages	828-839
Author	Wan Y Wang D Shewry PR Halford NG
Abstract	Summary: Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter. Three Ae. cylindrica and two T. timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined. A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae. cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T. timopheevi HMW subunits as 1Gx, 1Ax and 1Ay. It was not clear whether or not a 1Gy HMW subunit was present in T. timopheevi. The PCR products from Ae. cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T. timopheevi were derived from 1Ax and 1Ay genes. The 1Cx, 1Gx and 1Gy sequences were not amplified successfully. The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat (Triticum aestivum). Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high M(r) polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species. There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat. The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE. The truncated 1Dx gene from Ae. cylindrica failed to express in E. coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts
External Databases	http://link.springer.de/link/service/journals/00122/contents/01/00793/paper/s00122-001-0793-0.pdf
Keyword	[ Hide all but 1 of 58 ] abilities aegilops alignment amino acid amino acid sequence bread breadmaking quality breeding central repetitive domain chain codon cysteine cysteine residue d genome expressed protein gene glutenin hexaploid bread wheat high molecular weight high molecular weight subunits hmw subunit hmw subunits immunodetection isolation m-r making quality mobility molecular weight molecular weight subunits nucleotide nucleotide sequence pcr pcr products peptide polymer polymerase products protein protein fractions quality characteristics repeated sequences seed seed storage protein sequence sequencing silent similarities single seeds stop codon storage protein structure subunit subunit gene tritician triticum triticum aestivum triticum timopheevi weight subunits

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