Phytochrome is a biological switch that regulates gene expression in response to light, but the molecular basis of this regulation remains unknown. The objective of this proposal is to identify nucleotide sequences involved in phytochrome-regulated expression of Zea mays genes. The strategy involves isolation of cDNA clones for mRNAs whose transcription rates are rapidly regulated by phytochrome and use of these clones as probes to isolate the corresponding genomic DNA sequences for further analysis. Selection of the most rapidly regulated transcripts is designed to maximize the probability of obtaining genes that respond directly to the signal from the active Pfr form of phytochrome. In preliminary experiments we have identified a set of cDNA clones for transcripts that increase or a decrease in abundance within 1 hr from Pfr formation; have established a run-off transcription assay with isolated nuclei for determining whether the changes in mRNA abundance involve transcriptional regulation; and have isolated a genomic clone homologous to one of the cDNA clones. The proposed research will involve: (a) Isolation of additional cDNA clones to broaden the selection of sequences representing genes under rapid, coordinate phytochrome control; (b) Measurement of run-off transcription in isolated nuclei to determine which of these sequences are regulated at the transcriptional level in vivo; (c) Addition of purified phytochrome to isolated nuclei to test for Pfr-dependent, sequence-specific changes in transcription in vitro; (d) Isolation and sequencing of the 5'-flanking regions of genes homologous to the rapidly-regulated transcripts; and (e) Initiation of a search for phytochrome-responsive, regulatory sequences potentially present in the 5'-flanking DNA of these genes, by a combination of sequence comparison, DNA-binding protein assays, """"""""footprinting"""""""", and in vitro mutagenesis/gene transfer experiments. This research will contribute to an understanding of the molecular mechanism by which a central control system regulates plant gene expression in response to a major environmental stimulus. Data obtained with the readily manipulable phytochrome system are expected to be of importance in the more general context of efforts to define the molecular details of eukaryotic gene expression.
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