Hordein promoter methylation and transcriptional activity in wild-type and mutant barley endosperm
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- Sørensen, M.B., Müller, M., Skerritt, J. et al. Molec. Gen. Genet. (1996) 250: 750. doi:10.1007/BF02172987
B- and C-hordein gene transcription is severely reduced in the endosperm of the regulatory barley mutantlys3a, and this is correlated with persistent hypermethylation of the promoters. In contrast, D-hordein is expressed at normal levels in the mutant. To confirm the connection between methylation and transcriptional activity, a genomic D-hordein clone was isolated and sequenced. The nucleotide composition of the promoter region revealed a CpG island and methylation analysis, using bisulphite treatment of genomic DNA, confirmed that the D-hordein promoter is unmethylated in endosperm and leaf tissue. Immunocytochemical studies localized D-hordein to the reticular component of protein bodies in both the wild-type Bomi andlys3a. Transient expression ofGUS reporter gene constructs in barley endosperm, following transfection by particle bombardment revealed the D-hordein promoter to be 3–5 fold more active than B-or C-hordein promoters. Comparison of transient expression in Bomi andlys3a endosperm demonstrated that the activities of the unmethylated D-hordein and theHor1-14 C-hordein promoters were equivalent, while the activities in the mutant of theHor1-17 C-hordein and theHor2-4 B-hordein promoters were reduced two- and tenfold, respectively. Methylation of plasmids in vitro prior to expression severely inhibited B- and D-hordein promoter activities. Based on these observations two categories of promoters for endosperm-specific expression of storage proteins are recognized and a model involving methylation and modulation of chromatin structure in the regulation by theLys3 gene is presented.
Key wordslys3a CpG island Transient expression Particle bombardment Immunocytochemistry
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