Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.     

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweetpotato and its expression in response to stress

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens     

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants

The brown-midrib mutants of maize have a reddish-brown pigmentation of the leaf midrib and stalk pith, associated with lignified tissues. These mutants progressively became models for lignification genetics and biochemical studies in maize and grasses. Comparisons at silage maturity of bm1, bm2, bm3, bm4 plants highlighted their reduced lignin, but also illustrated the biochemical specificities of each mutant in p-coumarate, ferulate ester and etherified ferulate content, or syringyl/guaiacyl monomer ratio after thioacidolysis. Based on the current knowledge of the lignin pathway, and based on presently developed data and discussions, C3H and CCoAOMT activities are probably major hubs in controlling cell-wall lignification (and digestibility). It is also likely that ferulates arise via the CCoAOMT pathway.     

Helicoidal pattern in secondary cell walls and possible role of xylans in their construction

The helicoidal organization of secondary cell walls is overviewed from several examples. Both the plywood texture and the occurrence of characteristic defects strongly suggest that the wall ordering is relevant of a cholesteric liquid-crystal assembly that is rapidly and strongly consolidated by lignification. A preferential localization of glucuronoxylans, major matrix components, and in vitro re-association experiments emphasize their preeminent role: (1) during the construction of the composite as directing the cellulose microfibrils in a helicoidal array; (2) during the lignification of the composite as a host structure for lignin precursors.     

<Emphasis Type="Italic">Halobacillus locisalis</Emphasis> sp. nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea

A Gram-positive, motile, endospore-forming and rod-shaped halophilic bacterial strain MSS-155 (KCTC 3788 and KCCM 41687) was isolated from a marine solar saltern of the Yellow Sea in Korea and was subjected to a polyphasic taxonomic study. This organism grew at temperature of 10.0–42.0°C with an optimum of 35°C. Strain MSS-155 grew optimally in the presence of 10% NaCl and did not grow in the absence of NaCl. The cell wall peptidoglycan type of strain MSS-155 was A4 based on l-Orn-d-Asp. Strain MSS-155 was also characterized chemotaxonomically by having menaquinone-7 (MK-7) as the predominant isoprenoid quinone and anteiso-C_15:0 as the major fatty acid. The DNA G+C content was 44.0 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain MSS-155 falls within the radiation of the cluster comprising Halobacillus species. Levels of 16S rDNA sequence similarity between strain MSS-155 and the type strains of four Halobacillus species were in the range 97.6–98.8%. Strain MSS-155 exhibited levels of DNA-DNA relatedness of 6.2–11.2% to the type strains of Halobacillus species described previously. On the basis of phenotypic properties, phylogeny, and genomic data, strain MSS-155 should be placed in the genus Halobacillus as a member of a novel species, for which we propose the name Halobacillus locisalis sp. nov.Communicated by W.D. Grant     

A xylan-degrading strain of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>: isolation and characterization of the xylanase activity

Two strains (O and X₂) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O and X₂, grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100°C, and exhibited an optimal temperature and pH of 90°C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.Communicated by G. Antranikian     

Elemental and biochemical composition of<Emphasis Type="Italic"> Nephrops norvegicus</Emphasis> (Linnaeus 1758) larvae from the Mediterranean and Irish Seas

The Norway lobster, Nephrops norvegicus, is a commercially exploited decapod which is widely distributed throughout the north-eastern Atlantic and the Mediterranean Sea. Ovigerous females originating from the Mediterranean and the Irish Seas were held in the laboratory until larvae hatched. Biomass and biochemical composition, as well as digestive gland structure, were examined in newly hatched larvae from these two regions. In addition, previously published data from a North Sea population were included in our comparison. Elemental analyses showed that the absolute quantities of dry mass (DM), carbon (C), nitrogen (N) and hydrogen (H) (collectively referred to as CHN) per individual, and the C:N mass ratios, were significantly lower, while the relative CHN, protein and lipid values (in % of DM) were higher in samples from the Irish Sea compared to larvae originating from either the Mediterranean or the North Sea. As in CHN, the absolute level of protein per individual was higher in larvae from the Mediterranean, while no significant differences were observed in the individual lipid contents. Likewise, the digestive gland structure at hatching did not show any differences between study areas. Intraspecific variability in biomass and chemical composition of newly hatched larvae from different regions may be related to differential patterns of reproduction in regions with different climatic conditions. Lobster larvae hatch in the Mediterranean Sea predominantly in winter when both water temperature and planktonic food availability are at a minimum, while hatching in the Irish Sea occurs under more favourable conditions in spring. Hence, significantly higher wet mass, dry mass and protein values in Mediterranean larvae may represent adaptive traits allowing for early posthatching survival and development under food-limited conditions in an oligotrophic environment.Communicated by H.-D. Franke     

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.     

Large-scale exploration of growth inhibition caused by overexpression of genomic fragments in Saccharomyces cerevisiae

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.