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  • 1
    ISSN: 1573-5028
    Keywords: cucumber ; light-regulated gene expression ; NADH-dependent hydroxypyruvate reductase ; organ-specific gene expression ; peroxisome ; photorespiration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The 5′- and 3′-flanking regions of HPRA, a cucumber gene that encodes hydroxypyruvate reductase, were evaluated for regulatory activity with respect to light responsiveness and organ specificity. To define the functional regions of the 5′-flanking region of HPRA, a series of deletions was generated and the remaining portions fused to the β-glucuronidase (GUS) reporter gene (uidA) containing a minimal 35S promoter truncated at −90. The region from −66 to +39 was found to be necessary for light-regulated expression of the uidA reporter gene, while the region from −382 to −67 was found to be necessary for its leaf-specific expression. Further deletion of the HPRA 5′ flanking region to −590 resulted in high levels of root expression, suggesting the presence of a negative regulatory element responsible for silencing root expression of the HPRA gene between −590 and −383. The 3′-flanking region of the HPRA gene downstream of the polyadenylation site contains several sequence motifs resembling regulatory elements present in the promoters of several light-responsive genes. An 823 bp portion of the HPRA 3′-flanking region containing these putative regulatory elements enhanced GUS expression in leaves when placed downstream of the uidA reporter gene in the forward orientation, but not in the reverse orientation. When placed 5′ of the −90 35S promoter, the 823 bp fragment enhanced slightly, independently of orientation, the root tip-specific expression pattern intrinsic to the −90 35S promoter, indicating that in some cases this region can act as a transcriptional enhancer.
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  • 2
    ISSN: 1573-5028
    Keywords: cucumber ; cytokinin-responsive ; DNA-binding proteins ; hydroxypyruvate reductase ; transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (−382 to −67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (−382 to −285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5′-AAATGACGAAAATGC-3′, that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5′-AAGATTGATTGAG-3′, of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.
    Type of Medium: Electronic Resource
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