Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA
We have recently completed our characterization of the rice phytochrome gene
and are currently investigating its mode of regulation. We have sequenced a 3.8
kb cDNA clone and 10 kb of a genomic clone encompassing the entire gene. This
gene consists of 6 exons separated by 5 introns, the largest intron being 2585
bp and residing in the middle of the 5' leader sequence. The second exon
encodes the major N-terminal domain, which is conserved amongst a number of
species and enclodes the photoactive part of the holoprotein, including the
chromophore binding site.
The 5' cap site, as well as the boundaries ofthe large intron were mapped using high specific activity ssDNA probes hybridized to RNA under aqueous conditions. The genomic sequence includes 2kb of upstream sequences. A tandem TATA box is present at -30 bp, but no strong CAAT box is present. A striking feqture of the upstream region is 45 bp of a TA repeat from -290 to -245. We have commenced studies on protein factors that specifically bind to the upstream region. After optimization of methods for preparing nuclear extracts from rice leves, we have identified (by DNAse I footprinting and gel retardation analysis) a factor that binds to two different sites centered at -230 and -1350 bp upstream of the 5' start site. Mutational competition analysis and some preliminary methylation interference experiments identify this factor as GT-1, a protein previously shown to bind the LRE of the pea rbcS-3A gene. This interesting interaction, as well as other factors are being investigated further.
We have also made about 25 different constructs which have been transferred to tobacco to determine whether the upstream region is active in transgenic tobacco. We have been successful in expressing rice phytochrome mRNA in transgenic tobacco under the control of the 35S promoter/enhancer and are currently studying the synthesis, turnover and assembly of the rice phytochrome protein in tobacco.
We have available for distribution to other laboratories: cDNA and genomic libraries for IR36, as well as methods for isolation of nuclei and preparation of nuclear extracts.