Inheritance and structure of foreign DNA in progenies of transgenic tobacco obtained by direct gene transfer

Summary One of the transformed tobacco plants obtained by direct DNA transformation possessed two marker genes, a chimeric aminoglycoside phosphotransferase and nopaline synthase genes. Selfed progenies of this plant (T3-d) showed stable inheritance of these two genes. The minimum size of foreign DNA integrated into tobacco genome was estimated to be 5.4 kbp. A deleted nopaline synthase gene co-existed with an intact gene. The linkage analysis indicated that two transformants, T1-b and T3-c, possessed foreign DNA inserted in different chromosomes or in different sites of the same chromosome that recombine freely.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Synthesis of sulfated derivatives of curdlan and their anti-HIV activity

Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N-sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO₃-pyridine complex in a heterogeneous phase. It was shown from ¹³C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure.     

Redox-coupled conformational alternations in cytochrome c(3) from D. vulgaris Miyazaki F on the basis of its reduced solution structure

Heteronuclear NMR spectroscopy was performed to determine the solution structure of (15)N-labeled ferrocytochrome c(3) from Desulfovibrio vulgaris Miyazaki F (DvMF). Although the folding of the reduced cytochrome c(3) in solution was similar to that of the oxidized one in the crystal structure, the region involving hemes 1 and 2 was different. The redox-coupled conformational change is consistent with the reported solution structure of D. vulgaris Hildenborough ferrocytochrome c(3), but is different from those of other cytochromes c(3). The former is homologous with DvMF cytochrome c(3) in amino acid sequence. Small displacements of hemes 1 and 2 relative to hemes 3 and 4 were observed. This observation is consistent with the unusual behavior of the 2(1)CH(3) signal of heme 3 reported previously. As shown by the (15)N relaxation parameters of the backbone, a region between hemes 1 and 2 has more flexibility than the other regions. The results of this work strongly suggest that the cooperative reduction of hemes 1 and 2 is based on the conformational changes of the C-13 propionate of heme 1 and the aromatic ring of Tyr43, and the interaction between His34 and His 35 through covalent and coordination bonds.     

Solution structure of a SRP19 binding domain in human SRP RNA

Assembly of the human signal recognition particle (SRP) requires SRP19 protein to bind to helices 6 and 8 of SRP RNA. In the present study, structure of a 29-mer RNA composing the SRP19 binding site in helix 6 was determined by NMR spectroscopy. The two A:C mismatches were continuously stacked to each other and formed wobble type A:C base pairs. The GGAG tetraloop in helix 6 was found to adopt a similar conformation to that of GNRA tetraloop, suggesting that these tetraloops are included in an extensive new motif GNRR. Compared with the crystal structure of helix 6 in complex with SRP19 determined previously, the GGAG tetraloop in the complex was found to adopt a similar conformation to the free form, although the loop structure becomes more open upon SRP19 binding. Thus, SRP19 is thought to recognize the overall fold of the GGAG loop.     

Classification of RNA structures based on hydrogen bond and base-base stacking patterns: application for NMR structures

A computational system, CSNA, for classifying RNA structures according to structural characters was developed. CSNA lists up all the hydrogen bonds and base-base stackings in the structures, and classifies the structures into sub-groups based on their patterns as the first step grouping. The frequency of each hydrogen bond or base-base stacking is calculated, the frequency score being defined as the sum of the frequency of existing hydrogen bonds or base-base stackings for each sub-group. Finally, the sub-groups are further classified into groups based on the frequency score defined in this study and the difference between the patterns. According to the frequency score, CSNA suggests a group that shares most frequently appearing hydrogen bonds and base-base stackings. CSNA was applied to the classification of the results of two individual simulated annealing calculations based on NMR information. It was found that CSNA could extract structures with lower energy without checking any energy term and could provide well converged groups as the lowest energy structures. Thus, CSNA could be a new tool for structural determination of nucleic acids.     

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M₃ muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M₃ mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca²⁺ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands. translocation; aquaporin-5     

Interaction analysis between tmRNA and SmpB from Thermus thermophilus

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.