PubMed Health⌕ Search

Biomedical subjects

S A Kay

Publications and source records attributed to S A Kay.

71 records · Page 4Linked to original sources
PubMed1994

Circadian clock- and phytochrome-regulated transcription is conferred by a 78 bp cis-acting domain of the Arabidopsis CAB2 promoter.

The Arabidopsis CAB2 promoter was used to define the terminal genomic targets that are subject to regulation by the circadian clock. An in vivo cab::luciferase bioluminescent marker was used to enable the assaying of the expression of chimeric constructs with unprecedented sensitivity and time resolution in living seedlings. Dissection of -322 to +1 of the CAB2 promoter has revealed several interesting features: it was demonstrated that the 323 bp fragment contains at least one strong general positive element. The positive element contains an ACGT core sequence specifically bound by a protein activity, termed CUF-1, and contributes to high level expression but is not required for phytochrome- or circadian-regulation. Moreover, a 78 bp domain was defined that confers both circadian- and phytochrome-regulation upon heterologous promoters. Conserved GATA sequences within the 78 bp regulatory domain are specifically bound by a protein factor designated CGF-1. The binding specificity of CGF-1 appears to be related to the GT-family of trihelix DNA-binding proteins. The role of these DNA-protein interactions is discussed in terms of clock- and phytochrome regulation, and their relevance as targets for pathways defined by photomorphogenic mutants.

Arabidopsis↗
PubMed1993

Shedding light on clock controlled cab gene transcription in higher plants.

Research from several laboratories has revealed that the circadian clock regulates the transcription of many higher plant genes. Amongst the most well characterized of these genes are those that encode the chlorophyll a/b binding (Cab) proteins of the light harvesting complexes of photosystem II. Analysis of cab gene expression in both etiolated and green plants reveals a complex interaction between phytochrome(s) and the circadian clock, in which phytochrome appears to regulate the level of cab transcription and the circadian clock regulates the phase. The development of a non-invasive assay for cab transcription based on firefly luciferase expression in transgenic plants has not only made it possible to analyze clock regulated transcription in living plants, but also provides a means to screen for clock mutants in Arabidopsis.

Arabidopsis↗
PubMed1992

Characterization of a gene encoding a DNA binding protein with specificity for a light-responsive element.

The sequence element of box II (GTGTGGTTAATATG) is a regulatory component of a light-responsive element present within the upstream region of pea rbcS-3A. The nuclear protein GT-1 was defined previously as a DNA binding activity that interacts with box II. Here, we describe the isolation and characterization of cDNA sequences that encode a DNA binding protein with specificity for this element. The recombinant protein, tobacco GT-1a, shows similar sequence requirements for DNA binding to nuclear GT-1, as assayed by its ability to interact with previously defined 2-bp scanning mutations of box II, and is shown to be immunologically related to nuclear GT-1. The predicted structure of the 43-kD protein derived from the cDNA sequence suggests the presence of a novel helix-helix-turn-helix (HHTH) motif. Comparison between the predicted protein sequence encoded by the tobacco GT-1a cDNA and that of another GT binding protein, rice GT-2, reveals strong amino acid conservation over the HHTH region; this motif appears to be involved in the interaction between the recombinant protein and box II. Genomic DNA gel blot analysis indicated the presence of a small gene family of related sequences within the tobacco nuclear genome. RNA gel blot analysis of tobacco mRNA using the isolated cDNA as a probe showed that transcripts are present in several tissues, including both light-grown and dark-adapted leaves.

Amino Acid Sequence↗
PubMed1992

A novel circadian phenotype based on firefly luciferase expression in transgenic plants.

A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.

Animals↗
PubMed1991

Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings.

We have examined the biological activity of rice type I phytochrome (PI) in transgenic tobacco seedlings. The progeny of four independent transformants that expressed the rice PI gene segregated 3:1 for shorter hypocotyl length under dim white light (0.04 W/m2). By contrast, this phenotype was not observed either in the dark or under white light at higher intensity (6.0 W/m2). This suggests that the phenotype is dependent not only on light but also on light intensity. The increased light sensitivity cosegregated with the kanamycin-resistance marker as well as with the rice PI polypeptides, indicating that this phenotype is directly related to the expression of the transgene. The transgenic plants showing short hypocotyls exhibited a reduced growth rate throughout the elongation period, and the resulting shorter hypocotyl length was attributable to shorter epidermal cell length but not to reduced cell number. Furthermore, successive pulse irradiations with red light elicited short hypocotyls similar to those obtained under dim white light, and the effect was reversed by immediate far-red light treatment, providing a direct indication that the phenotype is caused by biologically active rice PI. Therefore, the far-red-absorbing form of the introduced rice PI appears to regulate the hypocotyl length of the transgenic tobacco plants through endogenous signal-transduction pathways. This assay system will be a powerful tool for testing the biological activity of introduced phytochrome molecules.

Journal Article↗
PubMed1990

Cloning and sequencing of protochlorophyllide reductase.

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.

Amino Acid Sequence↗
PubMed1990

Differential expression of two related organ-specific genes in pea.

We have screened a pea genomic library using a cDNA probe derived from pea shoot RNA. From this screen, we isolated two closely related genes, designated as S2 and P4. An intriguing property of these two genes is the presence in their coding region of a repeated sequence that is conserved between them in sequence but not in the number of the repeating units. The predicted amino acid sequence suggests that these proteins could be exported and glycosylated. 3' S1 analysis reveals that one of the genes, S2, is expressed highly in stem, as expected from previous work. However, mRNA derived from the other gene, P4, is not detectable in stem tissue, but is present in tissue derived from pea pods. The 5' upstream sequence of S2 and P4 are 94% identical up to position -121, suggesting that sequences upstream of -121 are responsible for organ-specific expression of the two genes.

Amino Acid Sequence↗
PubMed1989

The rice phytochrome gene: structure, autoregulated expression, and binding of GT-1 to a conserved site in the 5' upstream region.

We have isolated and characterized both cDNA and genomic clones encoding the apoprotein of rice phytochrome. The mRNA produced from this gene is expressed at a low level in etiolated leaves. Following a flash of red light, the steady-state mRNA level decreases within 15 minutes, and is barely detectable after 2 hours. This effect is partially reversed by far red light demonstrating autoregulation of phytochrome mRNA levels. Nuclear run-on experiments show that this effect is exerted on transcription of the phytochrome gene. In etiolated plants, phytochrome mRNA is twofold higher in leaves than in roots, whereas the reverse is true in fully green plants where phytochrome mRNA accumulates despite illumination of the leaves. DNA gel blots and screening of libraries indicate the presence of only a single gene, allowing convenient study of the autoregulatory phenomenon for a specific phytochrome gene. Gel retardation analysis using a fragment from the 5' upstream region reveals that GT-1 is present in nuclear extracts of etiolated rice leaves and binds to sites conserved between rice and oat phytochrome genes.

Base Sequence↗
PubMed1987

Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.

Pea nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor that specifically interacts with regulatory DNA sequences upstream of the pea rbcS-3A-gene. This factor, designated GT-1, binds to two short sequences (boxes II and III) in the -150 region that are known to function as light-responsive elements (LREs) in transgenic tobacco. Binding of GT-1 to homologous sequences further upstream (boxes II and III in the -220 region) indicates that these boxes comprise the redundant LRE that functions in vivo when boxes II and III are deleted. In both box II and box II, methylation interference experiments demonstrate that two adjacent G residues are critical for GT-binding. Single Gs present in boxes III and III are also important. Since GT-1 is present in nuclear extracts from leaves of light-grown and dark-adapted pea plants, its regulatory role does not depend on de novo synthesis. Thus if GT-1 binds differentially in vivo it must be postranslationally modified or sterically blocked from binding by another factor in response to light.

Base Sequence↗
PubMed1986

Phytochrome-controlled expression of a wheat Cab gene in transgenic tobacco seedlings.

We have monitored changes in the chlorophyll a/b-binding protein (Cab) mRNA levels in etiolated wheat leaves exposed to light of different wavelengths by Northern blot hybridizations and 5' S1 nuclease protection assays. Accumulation of the Cab mRNA and the specific Cab-1 transcript is regulated by phytochrome. Transcript levels are elevated by red light and the red enhancement can be partially reversed by far-red light. Moreover, far-red light alone can elicit a small increase in the transcript levels. These characteristic features of the wheat Cab-1 phytochrome response can be recapitulated in etiolated seedlings of transgenic tobacco containing the Cab-1 gene. Analyses of a chimeric construct revealed that a 1.8-kb 5'-flanking fragment (-1816 to +31) of the Cab-1 gene can confer phytochrome response as well as leaf-specific expression on a heterologous coding sequence.

Blotting, Northern↗
PubMed1983

Light-Induced Breakdown of NADPH-Protochlorophyllide Oxidoreductase In Vitro.

Light-induced loss of the enzyme protochlorophyllide reductase (EC 1.6.99.1.), already described as a characteristic of whole plants, has now been demonstrated in vitro using etioplast membrane preparations of Avena Sativa L. var Peniarth and Secale cereale L. var Rheidol. Some evidence is presented, based upon temperature, pH, and inhibitor sensitivity of the process, that loss of enzyme may be the result of proteolysis. The light-induced process can, in vitro, be largely prevented by addition of the substrates of the reductase, protochlorophyllide and NADPH. It is concluded that light causes the breakdown of the reductase in vivo and in vitro by producing ligand-free enzyme as a consequence of the photoconversion reaction.

Journal Article↗