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  • 1
    ISSN: 1573-5028
    Keywords: coordinate regulation ; Cucurbita phytochrome cDNA ; gene expression ; light regulation ; multiple transcripts ; regulatory photoreceptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated several cDNA clones for phytochrome from a dicot, Cucurbita pepo L. cv. Black Beauty (zucchini), and have used them to study the regulation of Cucurbita phytochrome mRNA levels. A cDNA library was constructed from poly(A)+ RNA isolated from etiolated Cucurbita hypocotyl hooks and enriched for phytochrome mRNA by size fractionation. This library was screened with a 32P-labeled fragment isolated from an Avena phytochrome cDNA clone. Several putative phytochrome clones were isolated and mapped by restriction endonuclease analysis. On the basis of this analysis there is no evidence for the expression of multiple phytochrome genes in Cucurbita. Recent sequence analysis has confirmed that the largest of these clones, pFMD1 (∼3.6 kb), does indeed encode phytochrome and that it contains the entire amino acid coding sequence for Cucurbita phytochrome (33). RNA blot analysis has revealed that two polyadenylated phytochrome transcripts (∼5.6 kb and ∼4.2 kb) are present in both cotyledons and hypocotyl hooks of Cucurbita. In etiolated Cucurbita seedlings given a saturating pulse of red light, the abundance of both transcripts coordinately declines to 50–60% of the dark levels within 3 h and reaccumulates to dark levels within 24 h. Reversal of induction of this response by a far-red light pulse immediately following red light treatment is not observed, which is in contrast to the far-red reversibility of the red light promoted decrease in phytochrome mRNA abundance observed in Avena (6). Etiolated seedlings transferred to continuous white light also show a coordinate decrease in the levels of the two RNAs to ∼40% of the dark levels within 3 h. The magnitude of the light-induced decline in phytochrome mRNA abundance in Cucurbita is substantially less than the decrease previously reported for Avena (6).
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: circadian regulation ; enzyme activity ; gene expression ; light regulation ; nitrate reductase ; phytochrome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of a number of plant genes is regulated by an endogenous circadian clock. We report that the Arabidopsis NIA2 (nitrate reductase) gene shows robust circadian oscillations in mRNA accumulation which persist for at least 5 days in plants that have been grown in a light-dark (LD) cycle and then transferred to continuous light (LL). We further show that NIA2 mRNA accumulation oscillates in a circadian fashion in plants that have been grown in LD and then transferred to continuous darkness (DD). Results from nuclear run-on transcriptional analysis suggest that the oscillations in steady-state levels of NIA2 mRNA abundance are not primarily due to changes in transcription but, instead, reflect post-transcriptional regulation. The circadian oscillations in NIA2 mRNA abundance are paralleled by circadian oscillations in nitrate reductase enzyme activity (NR activity) in Arabidopsis plants that have been grown in LD and then transferred either to DD or to LL. Etiolated Arabidopsis seedlings express neither NIA2 mRNA nor NR activity. However, both NIA2 mRNA accumulation and NR activity are induced by exposure to white light. The inductive effects of light on NIA2 mRNA accumulation are due, at least in part, to a very low fluence phytochrome-mediated response. However, the persistence of circadian oscillations in NIA2 mRNA abundance for at least 5 days in LL demonstrates that the circadian clock is capable of overriding or gating the inductive effects of light on NIA2 mRNA accumulation in Arabidopsis for an extended, continuous period of time.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-9368
    Keywords: gene expression ; transgenic monocots ; ubiquitin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A set of plasmids has been constructed utilizing the promoter, 5′ untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-β-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for bothUbi-GUS andUbi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice onBam HI andBam HI-compatible restriction fragments downstream of theUbi-1 gene fragment. Because theUbi-1 promotor has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 5 (1985), S. 91-101 
    ISSN: 1573-5028
    Keywords: gene expression ; low-abundance mRNA ; mRNA quantitation ; rapid regulation ; regulatory photoreceptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Pure phytochrome RNA sequence synthesized in an SP6-derived in vitro transcription system has been used as a standard to quantitate phytochrome mRNA abundance in Avena seedlings using a filter hybridization assay. In 4-day-old etiolated Avena seedlings phytochrome mRNA represents ∼0.1% of the total poly(A)+ RNA. Irradiation of such seedlings with a saturating red-light pulse or continuous white light induces a decline in this mRNA that is detectable within 30 min and results in a 50% reduction by ∼60 min and 〉90% reduction within 5 h. The effect of the red-light pulse is reversed, approximately to the level of the far-red control, by an immediately subsequent far-red pulse. In seedlings maintained in extended darkness after the red-light pulse, the initial rapid decline in phytochrome mRNA level is followed by a slower reaccumulation such that 50–60% of the initial abundance is reached by 48 h. White-light grown seedlings transferred to darkness exhibit a similar accumulation of phytochrome mRNA that is accelerated by removal of residual Pfr with a far-red light pulse at the start of the dark period. The data establish that previously reported phytochrome-regulated changes in translatable phytochrome mRNA levels result from changes in the physical abundance of this mRNA rather than from altered translatability.
    Type of Medium: Electronic Resource
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