Transcription of Arabidopsis and wheat Cab genes in single tobacco transgenic seedlings exhibits independent rhythms in a developmentally regulated fashion.

Transcription of Cab genes has been previously shown to be regulated by a circadian oscillator coupled to the red light-absorbing plant photoreceptor phytochrome in various plant species. In addition, it has recently been suggested that rhythmic expression of the Cab genes could also be affected by a phytochrome-independent circadian oscillator in a developmentally regulated fashion. This study has shown that a red light-insensitive oscillator and a phytochrome-coupled circadian clock indeed coregulate the oscillating expression of individual Cab genes at the level of transcription at an early developmental stage. The study involved analysing the expression patterns of transgenes, containing short fragments of the Arabidopsis thaliana Cab2 or the wheat Cab-1 promoter fused to the firefly luciferase reporter gene, by a video-imaging system in single, etiolated tobacco seedlings. Germination and red/far-red light treatments applied between 12 and 36 h after sowing lead to the appearance of two independent circadian rhythms. These rhythms coexist, both exhibiting period lengths close to 25 h but phased differently. However, repeated red-light treatments given 60 h or later after sowing synchronize these free-running rhythms and induce a single new circadian oscillation. These data indicate that both oscillators regulate the expression of the Cab genes studied at the level of transcription and that the cis-acting element(s) of the wheat Cab-1 and A. thaliana Cab2 genes mediating these responses are located on short, 250 bp promoter regions. Furthermore, these red-light induced rhythms are also inducible by far-red light treatments alone. Therefore, in tobacco, the phytochrome-coupled oscillator is regulated, at least partially, by the very low fluence response of phytochrome A.

Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis.

Ran, a small soluble GTP-binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells. Genes encoding Ran-like proteins have been isolated from different higher plant species. Overexpression of plant Ran cDNAs, similarly to their mammalian/yeast homologues, suppresses the phenotype of the pim46-1 cell cycle mutant in yeast cells. The mammalian/yeast Ran proteins have been shown to interact with a battery of Ran-binding proteins, including the guanidine nucleotide exchange factor RCC1, the GTPase-activating Ran-GAP, nucleoporins and other Ran-binding proteins (RanBPs) specific for Ran-GTP. Here, the characterization of the first Ran-binding proteins from higher plants is reported. The yeast two-hybrid system was used to isolate cDNA clones encoding proteins of approximately 28 kDa (At-RanBP1a, At-RanBP1b) that interact with the GTP-bound forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana. The deduced amino acid sequences of the At-RanBP1s display high similarity (60%) to mammalian/yeast RanBP1 proteins and contain the characteristic Ran-binding domains. Furthermore, interaction of the plant Ran and RanBP1 proteins, is shown to require the acidic C-terminal domain (-DEDDDL) of Ran proteins in addition to the presence of an intact Ran-binding domain. In whole cell extracts, the GST-RanBP1a fusion protein binds specifically to GTP-Ran and will not interact with Rab/Ypt-type small GTP-binding proteins. Finally, in good agreement with their proposed biological function, the At-Ran and the At-RanBP genes are expressed coordinately and show the highest level of expression in meristematic tissues.

The circadian oscillator is regulated by a very low fluence response of phytochrome in wheat.

Expression of genes encoding the light-harvesting chlorophyll a/b binding proteins of photosystem II (Cab) in etiolated wheat seedlings is controlled by phytochrome and a circadian clock. Even photoconversion of <1% of phytochrome to its active form, which can be achieved by moonlight, induces the expression of the Cab genes, particularly that of the Cab-1 gene, in circadian fashion. Thus, this reaction shows the characteristics of a low and a very low fluence response. A single far-red light pulse given to an etiolated seedling is sufficient for a persistence of the circadian oscillation of the Cab-1 mRNA level for at least 100 h. Subsequent red (R) or long-wavelength far-red (RG9) light irradiations alter the free running rhythm. These observations indicate a change in sensitivity to phytochrome and/or a control by stable phytochrome. The latter hypothesis is supported by the observation that the level of Cab-1 mRNA is increased or decreased by a second R or RG9 light pulse, respectively.

A 268 bp upstream sequence mediates the circadian clock-regulated transcription of the wheat Cab-1 gene in transgenic plants.

We previously reported that the expression of the wheat Cab-1 gene is regulated by an endogenous circadian rhythm and by the photoreceptor phytochrome both in wheat and in transgenic tobacco plants. To define regulatory elements necessary for the circadian rhythm-regulated Cab-1 gene expression, we now analysed the fluctuation of steady-state mRNA levels in a series of 5' deletion mutants in transgenic tobacco plants. We found that the expression of a deletion mutant containing 211 bp upstream sequence still exhibited circadian rhythm. Furthermore we show that an enhancer-like sequence of the Cab-1 promoter (from -357 to -90) can endow a chimaeric gene consisting of a truncated 35S promoter (from -90 to +8) and the bacterial beta-glucuronidase (GUS) gene with circadian clock-regulated gene expression. Finally we demonstrate by nuclear run-off experiments that the transcription rates of the Cab genes in wheat oscillate in a rhythmic manner, with a periodicity of approximately 24 hours. Consistent with our previous findings these results (i) indicate that the expression of the wheat Cab-1 gene is regulated mainly at the transcription level and (ii) identify a short promoter region between -211 and -90 that is responsible for the circadian clock-regulated gene expression.

Extensive homologous chloroplast DNA recombination in the pt14 Nicotiana somatic hybrid.

In a previous study, six recombination sites have been confirmed in the chloroplast DNA (cpDNA) of pt14, a somatic hybrid of Nicotiana tabacum and Nicotiana plumbaginifolia. In the present study, physical mapping revealed six recombination sites in the 11.4-kb SalI fragment alone, only one of which has been previously identified. This fragment is located in the large unique region. We assume, therefore, that the pt14 cpDNA is a fine mosaic of the parental genomes with a recombination site about every 2 kb. A 748-bp region that comprised the intergenic region between ORF73 and ORF74B, and 460 bp of the petD intron have been sequenced. Parent-specific sequences in the pt14 DNA defined the regions within which recombination took place. The exact site of recombination events could not be determined because the parental sequences were identical between the polymorphic markers, and these sequences have been preserved in the pt14 line.

Sequence and transcriptional analysis of a chloroplast insert in the mitochondrial genome of Zea mays.

The complete sequence of a mitochondrial DNA insertional event containing the 3' portion of the chloroplast 23S-4.5S rRNA gene, the entire 5S rRNA gene and intervening sequence and all but the 3' 6 nucleotides of the arginine tRNA gene is reported. Also reported are both chloroplast/mitochondrial DNA junction sequences, 551 nucleotides of flanking mitochondrial sequences and the genomic location of this insert in Zea mays mitochondria. Utilizing the distinctive transcriptional pattern seen for mitochondrial RNA derived from root tissue relative to shoot tissue, we also reported a general experimental test for whether chloroplast sequences transposed to the mitochondrion are transcribed. Although results for the insert reported suggest it is transcriptionally inactive, the technique should be generally applicable to any transposed sequence.

Interspecific chloroplast recombination in a Nicotiana somatic hybrid.

Genetic recombination between chloroplasts of two flowering plant species, Nicotiana tabacum and Nicotiana plumbaginifolia, after somatic cell fusion is described. The parental lines differed in three cytoplasmic genetic markers. The N. tabacum mutant SR1-A15 was streptomycin-resistant, defective in chloroplast greening, and lincomycin-sensitive. The N. plumbaginifolia mutant LR400 was streptomycin-sensitive, normal green, and lincomycin-resistant. Streptomycin-resistant clones in cell culture are identified by their ability to form a green callus on a selective medium. Streptomycin resistance in the SR1-A15 mutant could not be expressed due to defective chloroplasts. Protoplasts of the two species were fused, and calli grown from the fused population were screened for the expression of streptomycin resistance from the SR1-A15 line as the result of interspecific chloroplast recombination. A somatic hybrid, pt14, expressed a new combination of the cytoplasmic genetic markers. In the pt14 chloroplast genome three N. tabacum and four N. plumbaginifolia parent specific restriction sites have been identified, indicating that the pt14 chloroplast genome contains at least six recombination sites.

Purification and some properties of polynucleotide kinase from rat liver nuclei.

Polynucleotide kinase was purified from crude extracts of rat liver nuclei by affinity chromatography on DNA agarose. At optimal pH (5.5) and at saturating concentrations of ATP and DNA, the purified enzyme was found to express maximal activity in the presence of 0.10-0.15 M NaCl; higher salt concentrations inhibited the activity. At the optimal pH and NaCl concentration, the apparent KM for 5'-OH-DNA at 100 microM ATP was 46.2 microM and the apparent KM for ATP at 1 mM 5'-OH-DNA was 15.8 microM. Polynucleotide kinase was protected against heat inactivation by ATP as well as by 5'-OH-DNA at low and moderately high NaCl concentrations, which suggests that under these conditions the enzyme reacts according to a random reaction mechanism. Studies on the heat inactivation of the enzyme in the presence of 5'-OH- or 5'-P-DNA revealed the protection occurs only if 5'-OH-DNA is present, at NaCl concentrations permitting the enzyme to bind DNA.