Reference "Nucleotide sequence of barley chymotrypsin inhibitor-2 (CI-2) and its expression in normal and high-lysine barley"

Reference ID

8670

Title

Nucleotide sequence of barley chymotrypsin inhibitor-2 (CI-2) and its expression in normal and high-lysine barley

Source

European journal of biochemistry / FEBS, 1987, vol. 165, pp. 99-106

Authors (4)

Williamson-M-S	Forde-J
Buxton-B	Kreis-M

Abstract

cDNA clones for chymotrypsin inhibitor-2 (CI-2) have been isolated from an endosperm-
specific library of barley using a synthetic oligonucleotide probe. The
nucleotide sequences of several of the cDNAs predict an open reading frame
(beginning with an ATG codon) which encodes a protein of 84 residues (Mr 9380).
In the longest clone another ATG codon is present, a further 69 nucleotides
upstream. The nucleotide sequence between these two ATG codons predicts an amino
acid sequence with the characteristics of a signal peptide, as found in other
cloned plant protease inhibitors. However, it contains an in-frame TAA stop
codon, which is also present in all of the shorter cDNAs which extend into this
region. From in vitro translation experiments, using mRNAs synthesized from
cDNAs, we conclude that, in vitro, translation of all or the vast majority of
CI-2 mRNAs begins at the second ATG codon, 31 nucleotides downstream from the
ochre stop codon. Southern blotting of genomic DNA shows that CI-2 is encoded by
a small multigene family, while sequence analysis of the cDNAs shows that at
least two sub-families of mRNAs, which are more than 90% homologous, are present
in the endosperm. Northern blotting analysis shows that related but different
sequences are present in leaf and shoot RNA populations. Further Northern blot
hybridizations using RNA from the normal line, Sundance, and the high-lysine
barley mutant, Hiproly, show that endosperms of the latter contain greatly
increased levels of CI-2 mRNA. This correlates with the increased amount of CI-2
protein deposited in Hiproly, and demonstrates that the differential expression
of CI-2 in the two genotypes is controlled at the level of transcription and/or
stability of the mRNA. In contrast, the abundance of CI-2 mRNAs in leaves and
shoots is not affected.