Genetic variation at storage protein-coding loci of common wheat (cv ‘Chinese Spring’) induced by nitrosoethylurea and by the cultivation of immature embryos in vitro

Electrophoretic patterns of seed storage proteins, the high-molecular-weight glutenins and gliadins, were studied in 468 plants of the common wheat cultivar Chinese Spring regenerated from callus culture of immature embryos, in 115 plants grown from seeds treated with nitrosoethylurea and in 260 control plants. From 5 to 21 single grains were analysed from each plant. In these three groups, the frequency of inherited mutations causing the loss of all proteins controlled by a locus (null-mutations, probably caused by a chromosomal deficiency) was 0.69%, 2.07%, and 0.05% per locus (the differences were statistically significant), respectively, while that of mutations causing the loss of a single protein band was 0.11%, 0.33%, and 0.05%, respectively. The loss of all of the gliadins controlled by Gli-B1 or GH-B2 (mutations were probably caused by a deletion of satellites of the corresponding chromosomes), was significantly higher than the loss of gliadins controlled by genomes A and D. Gene mutations altering the electrophoretic mobility of a single protein band in the pattern were found only in the second group of plants (0.44%). Therefore, chemical mutagenesis which produced not only more mutations than cultivation of immature wheat embryos in vitro, but also a higher ratio of mutations that altered DNA sequences, can be considered as an easier and comparatively more promising way for obtaining new improved variants of loci controlling biochemical characteristics in wheat. Somaclonal variation, on the other hand, was probably mainly caused by chromosomal abnormalities and could therefore hardly be considered as a useful tool in wheat breeding.     

Pollen fertility restoration by nuclear gene Fr in CMS common bean: an Fr linkage map and the mode of Fr action

The Fr gene in common bean, Phaseolus vulgaris L., is a unique gene for the study of plant nuclear-mitochondrial interactions because it appears to directly influence plant mitochondrial genome structure, resulting in the restoration of pollen fertility in cytoplasmic male sterile plants. This gene action is distinct from other pollen fertility restoration systems characterized to date. As a first step towards the map-based cloning of this unusual nuclear gene, we identified RAPD markers linked to Fr using bulked segregant analysis of near-isogenic lines. Using DNA gel blot hybridization, we localized the identified RAPD markers to a linkage group on the common bean RFLP map and constructed a linkage map of the Fr region using both RAPD markers and RFLP markers. Analysis of the mode of Fr action with the aid of identified Fr-linked DNA markers indicated that Fr functions in a semidominant fashion, showing dosage effect in controlling the dynamics of a heteroplasmic mitochondrial population. We also present our observations on the developmental distinctions, crucial in the accurate mapping of the Fr gene, between spontaneous cytoplasmic reversion and Fr-driven fertility restoration, two phenomena that are phenotypically indistinguishable.     

Growth of an aerobic bacterium with trichloroacetic acid as the sole source of energy and carbon

The aerobic microbial decomposition of trichloroacetic acid (TCA) was studied. A TCA-decomposing culture was enriched in continuous-flow and batch experiments on a medium containing TCA as the only organic component. Pure cultures of TCA degraders were isolated from the enrichment on TCA agar plates. Characterization of several isolates proved them all to be representatives of the same bacterium, a Gram-negative, catalase-positive and cytochrome C-oxidase-positive, non-motile, somewhat irregular rod. The bacterium could not be identified on the basis of its carbon-source-utilization pattern, but a partial sequencing of the 16S rDNA gene showed the isolate to belong to the gamma sub-group of Proteobacteria, and to be phylogenetically close to Acinetobacter calcoaceticus. The isolated bacterium grew exponentially with TCA as the sole source of energy and carbon. The maximum growth rate (µ_max) and the growth yield on TCA (Y _X/S ) were determined to be 0.027 h^–1 and 0.027 g biomass/g TCA, respectively. The bacterium was not able to grow on mono- or dichloroacetic acid, but it could grow on acetate.     

Smoothed acyl chain orientational order parameter profiles in dimyristoylphosphatidylcholine-distearoylphosphatidylcholine mixtures: a 2H-NMR study.

The accommodation of chain-length mismatch in liquid crystal phase bilayers was examined by using deuterium nuclear magnetic resonance to obtain smoothed orientational order parameter profiles for acyl chains of both components in binary lipid mixture bilayers. Mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) covering a range of compositions were prepared with either DSPC acyl chains or DMPC acyl chains perdeuterated. Orientational order parameters in the plateau regions of the smoothed profiles for both components were found to increase smoothly with increasing DSPC concentration. The orientational order parameters in the DSPC-smoothed profile were found to be slightly higher than corresponding values for DMPC over a wide range of bilayer composition. The shapes of the smoothed profiles for both components were found to be sensitive to bilayer composition. At low DSPC concentration, DSPC methylene deuterons near the bilayer center display a secondary plateau at low orientational order. At high DSPC concentration, the plateau of the DMPC-smoothed profile is stretched slightly. The concentration dependence of the smoothed profiles at low DSPC concentration appears to be consistent with a picture in which the last few segments of the DSPC chain cross the bilayer midplane, on average, but remain very disordered.     

Influence of muscle activity on the forces in the femur: An in vivo study

Experiments were performed on two patients with custom-made instrumented massive proximal femoral prostheses implanted after tumour resection. In vivo axial forces transmitted along the prostheses were telemetered during level walking, single- and double-leg stance, and isometric exercises of the hip muscles. These activities varied the lever arms available to the external loads: minimum for double-leg stance and maximum for hip isometric exercises. Kinematic, force plate, EMG and telemetered force data were recorded simultaneously. The force magnification ration (FMR; the ratio of the telemetered axial force to the external force) was calculated. The FMRs ranged from 1.3 (during double-leg stance) to 29.8 (during abductors test), indicating that a major part of the axial force in the long bones is a response to muscle activity, the strength of which depends on the lever arms available to the external loads. From these results, it was shown that the bulk of the bending moment along limbs is transmitted by a combination of tensile forces in muscles and compressive forces in bones, so moments transmitted by the bones are smaller than the limb moments. It was concluded that appropriate simulation of muscle forces is important in experimental or theoretical studies of load transmission along bones.     

Neocentromere-mediated Chromosome Movement in Maize

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences. C urrent models suggest that chromosomes move by a combination of forces generated by microtubule disassembly (Inoue and Salmon, 1995; Waters et al., 1996) and the activity of molecular motors (Vernos and Karsenti, 1996; Yen and Schaar, 1996). Microtubule disassembly generates a constant poleward force; while molecular motors can generate force in either poleward or away-from-pole directions, depending on the characteristics of the motor protein. Both plus and minus end–directed microtubule-based motors are localized to kinetochores (Hyman and Mitchison, 1991). Immunolocalization experiments indicate that mammalian kinetochores contain the minus end– directed motor dynein throughout metaphase and anaphase (Pfarr et al., 1990; Steuer et al., 1990). The kinesin-like proteins CENP-E, which has a transient kinetochore localization in animals, and MCAK, which is localized between the kinetochore plates of mammalian chromosomes, are also thought to generate and/or regulate chromosome movement (Yen et al., 1992; Lombillo et al., 1995; Wordeman and Mitchison, 1995).In addition to the molecular motors on kinetochores, several kinesin-like proteins are localized to chromosome arms (Vernos and Karsenti, 1996). Two subfamilies of arm-based motors have been identified in animals: the NOD subfamily (Afshar et al., 1995; Tokai et al., 1996) and the Xklp1/chromokinesin subfamily (Vernos et al., 1995; Wang and Adler, 1995). Both Nod and Xklp1 are required for positioning chromosomes on the metaphase plate, suggesting that they encode plus end–directed motors (Afshar et al., 1995; Vernos et al., 1995). Other evidence suggests that minus end–directed motors interact with chromosome arms. In the plant Haemanthus, a poleward force acts along chromosome arms during metaphase (Khodjakov et al., 1996), and forces propelling chromosome arms poleward have been detected during anaphase in crane fly spermatocytes (Adames and Forer, 1996). Little is known about how poleward arm motility at metaphase–anaphase affects the fidelity or rate of chromosome segregation.The neocentromeres of maize (Rhoades and Vilkomerson, 1942) provide a particularly striking example of poleward chromosome arm motility. In the presence of Abnormal chromosome 10 (Ab10),¹ heterochromatic DNA domains known as knobs are transformed into neocentromeres and mobilized on the spindle (Rhoades and Vilkomerson, 1942; Peacock et al., 1981; Dawe and Cande, 1996). Knobs are primarily composed of a tandem 180-bp repeat (Peacock et al., 1981) which shows homology to a maize B centromere clone (Alfenito and Birchler, 1993). A characteristic feature of neocentromeres is that they arrive at the spindle poles in advance of centromeres; in extreme cases the neocentromere-bearing chromosome arms stretch towards the poles (Rhoades and Vilkomerson, 1942; Rhoades, 1952). A recently identified mutation (smd1) demonstrates that a trans-acting factor(s) encoded on Ab10 is essential for converting the normally quiescent heterochromatic knobs into active neocentromeres (Dawe and Cande, 1996).Here we use neocentromeres as a model for understanding the mechanisms and importance of nonkinetochore chromosome movement. As a part of our analysis, we developed a four-dimensional system for observing chromosome segregation in living meiocytes. Our experiments were designed to determine (a) how poleward arm motility affects the rate and fidelity of chromosome segregation; and (b) whether the mechanism of neocentromere motility is comparable to the mechanism of kinetochore motility.     

Molecular mapping of wheat. Homoeologous group 2.

A molecular-marker map of bread wheat having many markers in common with other grasses in the Gramineae family is a prerequisite for molecular level genetic studies and breeding in this crop species. We have constructed restriction fragment length polymorphism maps of the A-, B-, and D-genome chromosomes of homoeologous group 2 of hexaploid wheat (Triticum aestivum L. em. Thell) using 114 F7 lines from a synthetic x bread wheat cross and clones from 11 libraries. Chromosomes 2A, 2B, and 2D comprise 57, 60, and 56 markers and each spans about 200 cM. Comparisons between chromosomes are facilitated by 26 sets of homoeoloci. Genes mapped include a heterologous abscisic acid responsive locus cloned as pBS128, the epidermal waxiness inhibitor W21, and two presumed leaf rust and stem rust resistance genes. Anomalies suggesting ancestral rearrangements in chromosome 2B are pointed out and features of wheat group 2 chromosomes that are common to barley (Hordeum vulgare L.), rice (Oryza spp.), and T. tauschii are discussed.     

Purification and characterization of two essential cytochromes of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus A2 (Thiobacillus versutus)

Four c-type cytochromes were purified by several procedures including chromatography on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephadex G-75, G-100 and G-200 and chromatofocusing. Cytochrome c-551 had a pI value of 5.2 and an M_r of 260 000 consisting of six non-covalently bound polypeptides each with an M_r of 43 000, and contained four to five haems. Cytochrome c-552.5 had a pI value of 4.8 and an M_r of 56 000 consisting of two polypeptides with the same M_r 29 000, and contained two haems. Cytochromes c-551 and c-552.5 were reduced by ascorbate to about 70 and 60% of the fully dithionite-reduced values, respectively, and both were essential components in the thiosulphate-oxidizing multi-enzyme system (other components of the system were ‘enzyme A’, ‘enzyme B’ and sulphite: cytochrome c oxidoreductase). These two cytochromes functioned as electron carriers and effectors in the oxidation of thiosulphate. Some evidence suggested that cytochrome c-551 might be a specialized electron transfer component for sulphonate-sulphur oxidation. Both cytochromes could be reduced by thiosulphate in the presence of enzymes A and B. Cytochrome c-550 (basic) and cytochrome c-550 (acidic) were small proteins with M_r 15 000 and 14 000 and pI values of over 8 and 5, respectively. Their physiological role is uncertain.