Daron A. Fincham

Salinity induced differences in growth, ion distribution and partitioning in barley between the cultivar Maythorpe and its derived mutant Golden Promise

Dry matter changes and ion partitioning in two near isogenic barley cultivars Maythorpe (relatively salt sensitive) and Golden Promise (relatively salt tolerant) were studied in response to increasing salinity. Although the growth of both cultivars was significantly reduced by exposure to NaCl, the effect was greater in Maythorpe, whilst Golden Promise maintained an increased ratio of young to old leaf blade. Golden Promise maintained significantly lower Na+ concentrations in young expanding tissues compared with Maythorpe. Partitioning of Cl− was evident in that both varieties maintained lower Cl− concentrations in mesophyll than in epidermal cells. Golden Promise maintained higher K+/Na+ and Ca2+/Na+ ratios in young leaf blade and young sheath tissues than Maythorpe when exposed to salt. Differences in ion partitioning and the maintenance of higher K+ and Ca2+ to Na+ ratios, especially in young growing and recently expanded tissues, would appear to be important mechanisms contributing to the improved salt tolerance of Golden Promise.

Cloning of a novel gene encoding a C2H2 zinc finger protein that alleviates sensitivity to abiotic stresses in Aspergillus nidulans

We report the cloning and sequencing of a DNA fragment encoding a putative C 2 H 2 zinc finger protein from Aspergillus nidulans. The gene was isolated by complementation cloning of a salt sensitive phenotype of the A. nidulans sltAl mutant. A 3.8 kb Pst I fragment that restored wild type salt tolerance contained one large open reading frame of 2202 bp. The predicted protein (StzA) from this reading frame comprises 698 amino acids and has three Zinc fingers along with a putative transcriptional activation domain rich in acidic amino acids. The corresponding sequence from a sltAl mutant contains a premature STOP codon resulting in loss of the putative transcriptional activator in the C-terminal region. The Zinc fingers show conserved motifs with a number of transcription factors including CreA from A. nidulans and the human Wilms tumour susceptibility protein WT-1.

Evidence for sltA1 as a salt-sensitive allele of the arginase gene (agaA) in the ascomycete Aspergillus nidulans

Abstract Strains of Aspergillus nidulans carrying the sltA1 mutation, conferring sensitivity to KCl and NaCl, also showed an arginine-sensitive phenotype whereby concentrations of the L-amino acid at or above 10 mM were toxic to growth. Sexual progeny of a cross between a sltA1 mutant and a wild-type strain showed a co-segregation of salt and arginine sensitivity. Similarly, revertants to salt tolerance showed a loss of arginine sensitivity as did sltA1 strains that were transformed with a cosmid carrying the putative sltA1+ wild-type allele. In addition, arginine sensitivity could be relieved by L-ornithine. It is suggested that sltA1 is a salt-sensitive allele of the arginase gene (agaA).

Cation and harmaline interactions with Na+-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti)

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.

Complementation cloning of salt tolerance determinants from the marine hyphomycete Dendryphiella salina in Aspergillus nidulans

A preliminary physiological characterization of osmoregulation in an Aspergillus nidulans salt sensitive mutant carrying cotransformed DNA sequences from the marine hyphomycete Dendryphiella salina is described. A genomic DNA library made in lambda EMBL3 was cotransformed with the plasmid pDJB3 into an A. nidulans host carrying a selectable marker for transformation (pyrG89) and a salt sensitivity mutation (sltA1). In some cases D. salina clones appeared to directly complement the salt sensitivity mutation by restoring salt and arginine insensitivity. Other clones gave marked increases in salt tolerance and yet the hosts remained arginine sensitive. Improvements in salt tolerance correlated with an enhanced ability to induce polyol biosynthesis during salt challenge.

Nucleoside uptake by red blood cells from a primitive vertebrate, the pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Red blood cells from the Pacific hagfish (Eptatretus stouti) were found to possess a facilitated diffusion nucleoside transport system insensitive to inhibition by the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). Uridine uptake by this route was saturable (apparent Km 0.14 mM; Vmax 2 mmol/l cells per h at 10 degrees C), inhibited by inosine and adenosine, and blocked both by the vasodilator dipyridamole and by the thiol-reactive agent p-chloromercuriphenylsulphonate. The properties of this carrier resemble closely those of NBMPR-insensitive nucleoside transport systems in some mammalian neoplastic cell lines and in rat red cells. The presence of this type of carrier in a primitive vertebrate suggests that such transporters have a broad biological distribution and that they pre-date or arose at an early stage of vertebrate evolution.

Isotropic growth of spores and salt tolerance in Aspergillus nidulans

We report an investigation into the relationship between Aspergillus nidulans mutations sltA1 and agaA50 conferring salt sensitivity and arginase deficiency, respectively, and the ability of spores to swell and commence germination. Spores from both mutant strains exhibited uncontrolled swelling at pH 5.5 in the presence of arginine as the nitrogen source. The controlled swelling of spores from a salt tolerant strain under these conditions was disrupted by the Na+/H+ exchange inhibitor amiloride. Swelling in the presence of amiloride was associated with an elevation in the concentration of arginine within the spores of each strain tested. The possible consequences of alterations in the arginine economy of the spore for colony establishment in osmotically challenged habitats are discussed.

Assigning Level in Data-Mining Exercises.

Abstract There is currently much interest in ascribing outcomes to Masters (M) level programmes. It is particularly difficult to define M level outcomes in bioinformatics for students on non-specialist programmes. An approach is described that attempts to discriminate undergraduate from M level in a data-mining exercise. Differentiation of level is based upon the taxonomic origin of a DNA sequence, the relative increase in gene complexity from lower to higher eukaryote and the initiative required to use a wider range of databases and analytical tools.