A 43-kDa nuclear tobacco protein interacts with a specific single-stranded DNA sequence from the 5'-upstream region of the Agrobacterium rhizogenes rolC gene.

We identified in the nuclear extract of tobacco seedlings a 43-kDa DNA-binding protein RCS2 that interacted with the 5'-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid. DNA-protein gel shift and competition assays demonstrated that RCS2 bound to single-stranded DNA in a sequence-specific manner. A five-base direct repeat is important for the DNA binding of RCS2. South-Western blot analysis was employed to determine the size of RCS2 which appears to be approx. 43 kDa.

Cloning of a cDNA encoding an importin-alpha and down-regulation of the gene by light in rice leaves.

The import of nuclear proteins into nuclei begins with recognition of nuclear localization signal-harboring proteins and binding to a nuclear pore targeting complex. A cDNA for an importin-alpha protein, a subunit of the complex, was isolated from rice plants. The amino acid sequence deduced from the nucleotide sequence of the cDNA exhibited a high homology to those of importin-alpha proteins from many organisms such as Arabidopsis thaliana, Saccharomyces cerevisiae, human, mouse, Xenopus laevis and Drosophila melanogaster. Down-regulation of the transcription by light was shown in the leaves of light- and dark-grown seedlings by RNA blot analysis. The down-regulation was specific to leaves, whereas no light effect was observed in root tissues or calli, in which higher levels of the transcript were detected.

In vitro characterization of rice importin beta1: molecular interaction with nuclear transport factors and mediation of nuclear protein import.

We recently isolated two cDNAs encoding importin 3 homologues (rice importin beta1 and beta2), the first such homologues identified in plants. To address the function of rice importin beta1 in the process of nuclear import of proteins, we carried out in vitro binding and nuclear import assays. Recombinant protein of rice importin beta1 assembled a complex (PTAC) with rice importin alpha1 and NLS protein, and also bound to the nuclear envelope of tobacco BY-2 cells. Ran-GTP, but not Ran-GDP, interacted with rice importin beta1 and dissociated the heterodimer formed between rice importin alpha1 and rice importin beta1. An in vitro nuclear import assay using digitonin-permeabilized HeLa cells revealed that rice importin beta1 can mediate nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data strongly suggest that rice importin beta1 functions as a component of the NLS receptor in plant cells.

A novel importin alpha from rice, a component involved in the process of nuclear protein transport.

In eukaryotes, nuclear proteins that are transported into nuclei have nuclear localization signals (NLSs), which are recognized by proteins called importin alpha. We isolated a rice cDNA, #61L, and the corresponding gene that encodes a protein, which shows significant homology to the importin alpha. Although the encoded protein had only 23-27% amino acid identity to the importin alphas from various organisms including plants, the fusion protein with glutathione S-transferase showed a specific binding activity to the NLS of SV40 T-antigen. These results suggest that the rice #61L protein is a novel importin alpha in plants.

Production of transgenic gentian plants by particle bombardment of suspension-culture cells.

Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for ß-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l-1 hygromycin in liquid MS medium that contained 10 mg l-1 N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l-1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l-1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l-1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA.

New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus.

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.

Functional characterization of a plant importin alpha homologue. Nuclear localization signal (NLS)-selective binding and mediation of nuclear import of nls proteins in vitro.

Nuclear import of most nuclear proteins is initiated by recognition of the nuclear localization signal (NLS) by importin alpha. We recently isolated an importin alpha homologue from rice (rice importin alpha1) and demonstrated that transcription of the gene is down-regulated by light in rice leaves. To address the function of rice importin alpha1 in the process of nuclear import of proteins, we performed in vitro binding and nuclear import assays. The rice importin alpha1 showed specific binding to fusion proteins containing either monopartite or bipartite NLSs, but not to a fusion protein containing a Matalpha-2-type NLS, suggesting that there exists selective binding of rice importin alpha1 to different plant NLSs. The rice importin alpha1 is also capable of forming a complex with mouse importin beta and NLS protein in vitro. An in vitro nuclear import assay using permeabilized HeLa cells revealed that rice importin alpha1, in conjunction with other vertebrate transport factors, mediates the nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data provide strong, direct evidence suggesting that rice importin alpha1 functions as a component of the NLS receptor in plant cells.

Isolation and characterization of two importin-beta genes from rice.

The nuclear transport of proteins is mediated by the complex of importin-alpha and importin-beta. We isolated two cDNAs encoding importin-beta from rice. A rice importin-beta was demonstrated to interact with rice GST-importin-alpha fusion proteins. The presence of two importin-beta genes was shown for the first time among a variety of eukaryotes.

Detection of mutations in adenine phosphoribosyltransferase (APRT) deficiency using the LightCycler system.

We have applied an established technique, the polymerase chain reaction (PCR) with LightCycler technology, to a single disease with well-defined mutations. This assay produces results within only 30 min by combining PCR and fluorescence detection in one tube without electrophoretic band detection. In this study, we found 2,8-dihydroxyadenine (DHA) lithiasis in Japanese patients who were heterozygous for Japanese-type (type II) adenine phosphoribosyltransferase (APRT) deficiency (APRT*J). These patients, from a family with 2,8-DHA lithiasis, had a heterozygous mutation in the J region of the APRT gene. We demonstrated that the present system, using LightCycler technology, was simple, rapid, and reliable for detecting known mutations, and capable of identifying heterozygous and homozygous mutations in this family with APRT deficiency.

Phytochrome-mediated control of COP1 gene expression in rice plants.

We isolated a COP1 cDNA from rice and found that it could complement the Arabidopsis cop1-4 mutant. The putative rice COP1 protein has the Ring-finger, coiled-coil. and WD-40 repeat domains, which are also conserved in pea, tomato, and mammalian COP1 proteins. The degree of overall identity between rice COP1 and Arabidopsis COP1 is 73%, and the similarity value is 83%. Expression of rice COP1 was detected in almost all plant tissues, with the level being relatively higher in calli and very low in etiolated leaves. The expression level was positively controlled by light in etiolated and green leaves. At the end of the light period, expression of the gene in green leaves could be down-regulated by far-red light. This far-red light effect could be prevented by subsequent irradiation with red light. These results indicate that phytochrome regulates rice COP1 expression.

Molecular cloning of a novel importin alpha homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported.

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin alpha, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin alpha homologue from rice which is in addition to our previously isolated rice importin alpha1a and alpha2, and we have named it rice importin alpha1b. In vitro binding and nuclear import assays using recombinant importin alpha1b protein demonstrate that rice importin alpha1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin alpha genes revealed that the genes were not only differentially expressed but that they also responded to dark-adaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin alpha1b. These data suggest that each of the different rice importin alpha proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin alpha contribute to the regulation of plant nuclear protein transport.