Isolation,characterization and application of a species-specific repeated sequence from Haynaldia villosa

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.     

RFLP tagging of a salt tolerance gene in rice

A salt tolerant rice mutant (M-20) was obtained through selection in vitro. Its tolerance was stably inherited over eight generations and most traints between M-20 and its sensitive original 77–170 (Oryza sativa) were very similar. By deriving an F₂ population of M-20 × 77–170 and splitting every F₂ individual into two parts, with one part planted in normal conditions and another part in saline conditions, the inheritance of salt tolerance in rice was studied. Under normal conditions, there was no apparent segregation among F₂ individuals. Under saline conditions, however, the segregation of traits was obvious. According to our standards, the ratio of salt sensitive:moderately-tolerant:tolerant plants was 25:42:18, in accordance with a 1:2:1 ratio. It suggested that the improvement of salt tolerance in our materials was induced by the mutation of a major tolerant gene which showed incomplete dominance. By use of 130 RFLP probes distributed throughout the rice genome, the gene was tagged by a single copy DNA probe, RG4, which was located on chromosome 7. The genetic distance between the salt tolerant gene and RG4 was 7.0 ± 2.9 cM. Based on the split method, a method which could be currently used to evaluate the damage of salt stress in rice was proposed.     

An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.     

Inputs from upper airway affect firing of respiratory-associated midbrain neurons

Chen, Zibin, and Frederic L. Eldridge. Inputs fromupper airway affect firing of respiratory-associated midbrain neurons. J. Appl. Physiol. 83(1): 196-203, 1997.In 16 decerebrated unanesthetized cats, we studied effects ofneural inputs from upper airway on firing of 62 mesencephalic neuronsthat also developed respiratory-associated (RA) rhythmic firing whenrespiratory drive was high [Z. Chen, F. L. Eldridge, and P.G.Wagner. J. Physiol. (Lond.) 437:305-325, 1991] and on firing of 16 neurons that did notdevelop the rhythmic firing (non-RA neurons). Activity in RA neuronsincreased after mechanical expansion of pharynx (45% of those tested)or larynx (68%) and after stimulation of glossopharyngeal (50%) orsuperior laryngeal nerves (77%). The increased neuronal firingoccurred despite decreases or abolition of respiratory activity(expressed in phrenic nerve). Neuronal firing also increased aftermechanical stimulation of nasal mucosa (66%) or by jetsof air directed into the nares (48%) and after lightbrushing of nasal skin (~40%). Most stimuli led to decreased firingin a smaller number of neurons, and some neurons showed no response.None of the non-RA neurons developed an increase of firing after any ofthe stimuli, although one had decreased firing after stimulation of thesuperior laryngeal nerve. We conclude that inputs from the upper airwayand nasal skin have independent modulatory effects on the samemesencephalic neurons that are stimulated by ascending rhythmic RAinput from the medulla. These findings may have relevance to generationof the sensation of dyspnea.

     

Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria

Rhodamine 123 accumulates in the mitochondria of living cells and exhibits selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the F0F1 ATPase complex and possibly electron transfer reactions as well.     

Distribution of cell surface lets protein in co-cultures of normal and transformed cells

When LETS protein positive and negative cells were co-cultured, the positive cells remained as positive and the negative cells remained as negative. Apparently the transformed cells do not secrete factors which are sufficient to influence the distribution of surface LETS protein on normal cells.     

High performance liquid chromatography of pharmacologically active amines and peptides in biological materials

Precise and quantitative reversed-phase high performance liquid chromatographic (HPLC) procedures are described which can be used in biogenic amine and neuropeptide research. The amine procedure was applied to various pharmacological matrices including plasma, heart tissue and brain. The use of peptide HPLC as an analytical tool for various neuropeptides is illustrated by studies on des-tyrosine-gamma-endorphin (DT gamma E) metabolism in the brain and the stability of an ACTH (ORG-2766) analogue during a chronic infusion in rats. The power of HPLC as a research tool in peptide pharmacology is described, discussed and demonstrated as an aid in the understanding of the pharmacological effects of exogenous peptides and the function of the brain.     

Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells

A reduce uptake and retention of the mitochondria-specific membrane potential probe rhodamine 123 by feline sarcoma virus (FeSV)-transformed mink fibroblasts (64F3) has been detected. The decreased accumulation of rhodamine 123 by 64F3 mitochondria is not due to abnormal plasma membrane dye permeability, since after microinjection of the dye these cells are still unable to retain the dye at levels comparable to the untransformed parental cells, CCL 64. Nigericin, an ionophore that mediates an electrically neutral exchange of protons for potassium ions resulting the elimination of the pH gradient across the mitochondrial membrane and a compensatory increase in mitochondrial membrane potential with continued respiration, increases both the dye uptake and the retention time in transformed 64F3 cells. These results suggest that mitochondria in FeSV-transformed mink cells may have an abnormally low mitochondrial membrane potential accompanied by a relatively high pH gradient. Since anioic metabolites such as pyruvate and glutamate are accumulated by mitochondria in proportion to the delta pH across the mitochondrial membrane, the abnormal mitochondria described here may contribute to the abnormal metabolic state of FeSV-transformed cells.     

Computer simulations of mass transport for nonidentical interacting molecules.

We have developed a system for simulating the mass transport properties of interacting nonidentical macromolecules where the association is of the form A + B ? C, C + B ? D. Our simulation programs operate in a minicomputer (PDP 1104) with 16K of core and provide results identical to methods previously usable only with large computers. We use a rectangular approximation for the centrifuge cell which greatly simplifies calculation, although it introduces a few percent error into any attempt at quantitative fitting of actual data. The program as written is directly applicable to gel chromatography, simply by substitution of flow for centrifugal field and dispersion for diffusion. Simulations of centrifuge results have been compared with experimental results for two systems which have been proposed to fit the association pattern described—nitrogenase components and an antigen-antibody interaction. In both cases the results of our simulations suggest that the accepted interpretation of the experimental results may need to be modified. For the antigen-antibody interaction, the presence of multivalent higher order complexes apparently is required to explain the centrifuge results. For nitrogenase, one cannot readily distinguish the case of association to form both 1:1 and 1:2 molar complexes from that of formation of only the 1:1 complex on the basis of the published data. Criteria for making such a discrimination are discussed.