Isolation of a vicilin gene from pea (pisum sativum l.), and nuclease sensitivity of seed storage protein genes in pea chromatin

Abstract

A library of pea genomic DNA in the bacteriophage vector EMBL3 was screened by hybridisation to cDNAs encoding vicilin, a major storage protein of pea (Pisum sativum L.) seeds. A vicilin gene, vic A, was isolated and characterised by restriction mapping and DNA sequencing. The nucleotide and predicted amino acid sequences of vic A were compared to those of vicilin cDNAs, and the gene was found to encode a 50,000M(_r) non-glycosylated vicilin subunit that does not undergo post-translational proteolytic cleavage. The introns in vic A were typical of those in plant genes, being small and high in A+T content, and the nucleotide sequences at the splice sites showed good homology to the plant consensus. The positions of the introns in vic A were similar to those in a gene encoding a subunit of phaseolin, a related protein from French bean (Phaseolus vulgaris). Methods were developed for the analysis of nuclease sensitivity of specific genes in pea chromatin. The DNAase I sensitivity of the seed storage protein genes was found to be greater in developing cotyledons, where the genes were transcriptionally active, than in leaves, where they were inactive. The pea ribosomal genes showed relative resistance to DNAase I in both tissues. The nucleosome repeat length, determined by digestion of chromatin with micrococcal nuclease, was similar in both tissues. No evidence was obtained for DNAase I hypersensitive sites in pea chromatin. This result supports the findings of two other studies, and suggests that such sites are absent from plant chromatin.