Induction and characterization of Ph1 wheat mutants

The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy. We present here one such strategy focusing on the homologous pairing suppressor (Ph1) locus of wheat. This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control. The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content. As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A). These loci are located within 400 kb of each other in the rice genome. By sequencing this region of the rice genome, it should now be possible to define the nature of this factor.     

Distribution of Glutamate Receptors of the NMDA Subtype in Brains of Heterozygous and Homozygous Weaver Mutant Mice

In weaver mice, mutation of an G-protein inwardly rectifying K⁺ channel leads to a cerebellar developmental anomaly characterized by granule and Purkinje cell loss and, in addition, degeneration of dopaminergic neurons. To evaluate other deficits, glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) were examined by autoradiography with [³H]MK-801 in 36 brain regions from heterozygous (wv/+) and homozygous (wv/wv) weaver mutants, and compared to wild type (+/+) mice. In wv/+ and wv/wv mutants labelling decreased in cortical regions, septum, hippocampus, subiculum, neostriatum, nucleus accumbens, superior colliculus and in the cerebellar granular layer. The reductions in [³H]MK-801 binding were particularly specific in the cerebellar granular layer of wv/wv mutants, but an ubiquitous altered NMDA receptor topology was revealed in other brain regions. Abnormal developmental signals, or aberrant cellular responses, may underlie widespread NMDA receptor reductions, while in cerebellar cortex they could be lacking due to the massive loss of cerebellar granule cells.     

Nico M. van Gelder (1933–2005)

IN MEMORIAM

Nico M. van Gelder (1933–2005)     

Comparative genetic analysis of the Aegilops longissima and Ae. sharonensis genomes with common wheat

Aegilops longissima Schw. et Musch. (2n= 2x=14, S^lS^l) and Aegilops sharonensis Eig. (2n=2x=14, S^lS^l) are diploid species belonging to the section Sitopsis in the tribe Triticeae and potential donors of useful genes for wheat breeding. A comparative genetic map was constructed of the Ae. longissima genome, using RFLP probes with known location in wheat. A high degree of conserved colinearity was observed between the wild diploid and basic wheat genome, represented by the D genome of cultivated wheat. Chromosomes 1S^l, 2S^l, 3S^l, 5S^l and 6S^l are colinear with wheat chromosomes 1D, 2D, 3D, 5D and 6D, respectively. The analysis confirmed that chromosomes 4S^l and 7S^l are translocated relative to wheat. The short arms and major part of the long arms are homoeologous to most of wheat chromosomes 4D and 7D respectively, but the region corresponding to the distal segment of 7D was translocated from 7S^lL to the distal region of 4S^lL. The map and RFLP markers were then used to analyse the genomes and added chromosomes in a set of ’Chinese Spring’ (CS)/Ae. longissima chromosome additions. The study confirmed the availability of disomic CS/Ae. longissima addition lines for chromosomes 1S^l, 2S^l, 3S^l, 4S^l and 5S^l. An as yet unpublished set of Ae. sharonensis chromosome addition lines were also available for analysis. Due to the gametocidal nature of Ae. sharonensis chromosomes 2S^l and 4S^l, additions 1S^l, 3S^l, 5S^l, 6S^l and 7S^l were produced in a (4D)4S^l background, and 2S^l and 4S^l in a euploid wheat background. The analysis also confirmed that the 4/7 translocation found in Ae. longissima was not present in Ae. sharonensis although the two wild relatives of wheat are considered to be closely related. The phenotypes of the Ae. sharonensis addition lines are described in an Appendix. Received: 28 September 2000 / Accepted: 19 January 2001     

Shoot regeneration after fire or freezing temperatures and its relation to plant life-form for some heathland species

Shoot regeneration after prescribed burning or following the freezing temperatures of winter was monitored for nineteen heathland species present in an Arctostaphyleto-Callunetum community in northeast Scotland. Species whose renewal buds were near the surface of the ground started to grow earlier in the spring than species with renewal buds above the surface, but grouping species according to the position of their renewal bud (i.e. their life-form) did not account for all of the interspecific variation apparent. In the case of shoot regeneration after fire, species whose renewal buds were destroyed by fire because they were above-ground started to regenerate about the same time as species with belowground buds, protected from fire, but reached their maximum frequency of occurrence later. Grouping species by life-form was of limited value as a means of interpreting this interspecific variation in the timing of shoot regeneration after fire. It would be unwise to use plant life-form as the sole basis for interpreting or predicting a species' response to temperature stress when extreme temperatures occur regularly, as they do in heathland. The possible use of other plant traits to interpret and predict interspecific variation in the regeneration rate of heathland plants is discussed.Nomenclature follows Tutin et al. (1964–1980) for vascular plants. Acknowledgements. The Nature Conservancy Council and Mr J. J. Humphries kindly allowed Dinnet Moor to be used for the work presented here. One of us (RJR) received financial support for field work from the Natural Sciences and Engineering Research Council of Canada.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Evidence for the participation of disulfide and sulfhydril groups in the specific binding of [3H]prazosin in cerebral cortex

The tritiated ₁ antagonist prazosin [³H]PRZ binds specifically and with high affinity to postsynaptic adrenoceptors in membrane preparations from cerebral cortex. Since adrenoceptors are of protein nature, it was of interest of investigate the possible role of disulfide (—SS—) and sulfhydril (—SH) groups in the binding of [³H]PRZ. Pretreatment of the membranes with the disulfide and sulfhydryl reactivesdl0Dithiothreitol,l-Dithiothreitol, Dithioerythritol or 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), alone or in combination with the alkylating agent N-Methylmaleimide (NMM), decreased specific [³HPRZ binding, with minor changes in the non-specific counts. Saturation experiments revealed that all these reagents reduced the affinity of the binding site for [³H]PRZ, as judged by theK _d 25°C, but only the alkylating agent NMM and the oxydizing reagent DTNB produced in addition to the increase inK _d, a decrease of the maximum binding capacity (B _max). The present results provide evidence for a participation of—SS—and/or—SH groups in the recognition site of the ₁-adrenoceptor of cerebral cortex.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.