Hypoxanthine: Guanine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

Summary Yeast mutants lacking activity of the enzyme hypoxanthine: guanine phosphoribosyltransferase (H:GPRT) have been isolated by selecting for resistance to 8-azaguanine in a strain carrying the wild type allele, ade4 ⁺ of the gene coding for amidophosphoribosyltransferase (PRPPAT), the first enzyme of de novo purine synthesis. The mutants excrete purines and are cross-resistant to 8-azaadenine. They are recessive and represent a single complementation group, designated hpt1. Ade4-su, a prototrophic allele of ade4 with reduced activity of PRPPAT, is epistatic to hpt1, suppressing purine excretion and resistance to azaadenine but not resistance to azaguanine. The genotype ade2 hpt1 does not respond to hypoxanthine. Hpt1 complements and is not closely linked to the purine excreting mutants pur1 to pur5. Hpt1 and pur6, a regulatory mutant of PRPPAT, are also unlinked but do not complement, suggesting a protein-protein interaction between H:G-PRT and PRPPAT. Mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis, inhibits the growth of hpt1 and hpt1 ⁺. Xanthine allows both genotypes to grow in the presence of MPA whereas guanine only allows growth of hpt1 ⁺. Activity of A-PRT, X-PRT and H:G-PRT is present in hpt ⁺. Hpt1 lacks activity of H:G-PRT but has normal A-PRT and X-PRT.     

Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought

Potato plants ( Solanum tuberosum L. cvs 'Up-to-Date', 'Desiree', 'Alpha', 'Spunta', 'Elvira' and 'Troubadour') were exposed to cycles of water stress and relief during growth. Severe water deficit induced increased proline content 6- to 7-fold in nonturgid leaves which just started to wilt, and 8- to 27-fold in fully wilted leaves of potatoes. However, proline content was not affected during the early stages of stress development over a range of osmotic potentials in the leaves. The rising proline content was related to turgor loss of leaves independent of changes in the osmotic potentials, which indicates that proline involvement in osmoregulation of potato leaves is unlikely.
Repeated cycles of water stress and relief resulted in increased proline and α-amino nitrogen content in the tuber tissue of some of the cultivars. The smallest increase in proline content was obtained in 'Alpha' tubers and the content of α-amino nitrogen remained unaffected by the water stress. Concomitantly, 'Alpha' was the most drought-tolerant cultivar, as determined by its capacity to accumulate dry matter in tubers under stress conditions. On the other hand, in tubers of cultivars which were more susceptible to drought, a marked increase in proline and α-amino nitrogen was observed in response to water stress. The possible association of these findings with tolerance of potatoes to repeated short periods of drought is discussed.     

Physiological changes in, and phosphate uptake by potato plants during development of, and recovery from phosphate deficiency

The development of phosphate deficiency (P-stress) was observed in rooted sprouts of Solanum tuberosum L. cv. Desiree growing in solutions without phosphate. Shoot growth was inhibited by P-stress within 3 to 5 days of terminating the phosphate supply, while significant effects on root growth were not recorded until 7 to 9 days. Thus, the shoot:root dry weight ratio decreased from 4.3 to 2.6 over a 10-day period. Growth in the absence of an exogenous phosphate supply progressively diluted the phosphorus in the plant. The proportional decrease in concentration was similar in roots and shoots over a 7-day period, even though the former were growing more quickly. The potential for phosphate uptake per unit weight of root increased rapidly during the first 3 days of P-stress. When the plants were provided subsequently with a labelled, 1 mol m^?3 phosphate solution, the absorption rate was 3 to 4-fold greater than that of control plants which had received a continuous phosphate supply. The increased rate of uptake by P-stressed plants was accounted for by an increase (3-fold) in the V_max of system 1 for phosphate transport and by a marked increase in the affinity of the system for phosphate (decrease in K_m). In the early stages of P-stress, before marked changes in growth were measured, the proportion of labelled phosphate translocated to the shoots increased slightly relative to the controls when a phosphate supply was restored. In the later stages of stress a greater proportion was retained in the root system of P-stressed plants than in that of controls. In plants with roots divided between solutions containing or lacking a phosphate supply, the increased absorption rate was determined by the general demand for phosphate in the plant and not by the P-status of the particular root where uptake was measured. By contrast, the poportion translocated was strongly dependent on the P-status of the root. The restoration of a phosphate supply to P-stressed plants was marked by a rapid increase in the P concentration in snoots and roots which returned to levels similar to unstressed controls within 24 h. The enhanced uptake rate persisted for at least 5 days, resulting in supra-normal concentrations of P in both shoots and roots, and in the formation of extensive necrotic areas between the veins of mature leaves. Autoradiographs showed accumulations of ³²P in these lesions and at the points where guttation droplets formed on leaves.     

The regulation of glycolysis and electron transport in roots

The respiration of roots and isolated root mitochondria was investigated in Phaseolus vulgaris L., Spinacea oleracea L.; Triticum aestivum L., and Zea mays L. Although the respiration of both intact roots and isolated mitochondria displayed resistance to cyanide and sensitivity to SHAM, the percentage resistance and inhibition in roots was not the same as that in the mitochondria, with the exception of wheat. Adding FCCP to roots stimulated oxygen uptake and equalized the effects of SHAM and cyanide on roots and mitochondria. In spinach and maize roots, FCCP stimulated both the cytochrome and alternative pathways, while in bean roots, only the alternative pathway was stimulated. FCCP had little effect on wheat root respiration rates. Potential in vivo rates of oxygen uptake were estimated by expressing rates obtained with isolated mitochondria on a fumarase activity basis, and fumarase activity on a root weight basis. In wheat roots the potential rate was approximately equal to the measured in vivo rate; in the other species the potential rates were substantially greater than measured rates, but approximately equal to uncoupled in vivo rates. Key glycolytic intermediates in roots were measured, and it was found that the phosphofructokinase and pyruvate kinase reactions were displaced far from equilibrium, the degree of displacement being approximately equal in roots with little, and roots with substantial, alternative path engagement. Thus, although glycolysis is controlled, the regulation of this pathway appears to be quite flexible. The results are discussed in terms of possible regulatory mechanisms.     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

Thermodynamic approach to biomass distribution in ecological systems

In the derivation of the biomass distribution function for an ecological population critical use is made of an energetic constraint on the maximization of biomass diversity. The nature of this constraint is explored in detail using Kleiber's relation σ(m)=cm ^γ between animal metabolic rate σ(m) and body weightm in conjuction with the Prigogine-Wiame thermodynamic paradigm for specific entropy production in biological stationary states. These two inputs fix the energetic constraint on the maximization of biomass diversity to be the constancy of the mean metabolic rate of the ecosystem. The resulting biomass distribution function is tested against observational data.     

Spheroplast fusion as a mode of genetic recombination in mycobacteria

Spheroplasts were prepared from two carotenoid pigment mutants of Mycobacterium aurum named NgR9 and A11, which were obtained by the chemical mutagenesis of the wild type strain A+ with N-methyl-N'-nitro-N-nitrosoguanidine. The carotenoid pigments and the alpha- and beta-mycolic acids were taken as genetic markers and the recombinants were selected on the basis of their colour on L?wenstein-Jensen medium. Spheroplasts of the two mutants were mixed in a 1:1 ratio and were treated with 40% (w/v) polyethylene glycol 6000 for 5 min at 37 degrees C. The frequency of NgR9 X A11 recombination in optimal conditions was about 2.5 X 10(-3). The recombinants selected on the basis of their carotenoid pigment profile were also tested for their alpha- and beta-mycolic acids as a second genetic marker. The results were further confirmed by electron microscopy. The optimal conditions for spheroplast fusion as a mode of genetic recombination in M. aurum are described.     

Synthesis of cathepsin B by cells derived from the HL60 promyelocytic leukaemia cell line

Cells of the human promyelocytic HL60 line were induced to differentiate into granulocyte-like cells with dimethylsulphoxide (DMSO) or macrophage-like cells with 12-0-tetradecanoylphorbol-13-acetate (TPA). The synthesis of Cathepsin B by these cells was studied by immunoperoxidase staining and assay of cell lysates using the fluorimetric substrate benzoyloxycarbonyl-phenylanalyl-arginine-4-methyl-7-coumarylamide. Only 2–5% of the uninduced HL60 cells and DMSO-induced cells were immunohistochemically positive for Cathepsin B, compared with over 80% of the TPA-induced cells. Cathepsin B activity was lowest in the lysates of uninduced HL60s. DMSO-induced cells contained 1.5–2-fold the enzyme activity of HL60s and TPA-induced cell lysates demonstrated 5–14-fold the activity of uninduced HL60s. Induction of Cathepsin B synthesis was therefore associated with differentiation of the promyelocytes into cells of the monocyte/macrophage type, but not granulocyte-like cells. Cathepsin B was located immunohistochemically in human palatine tonsils. The enzyme was only demonstrated within macrophages in these tissues. Cathepsin B may therefore be a useful immunohistochemical marker for malignant and nonmalignant cells of the monocyte/macrophage lineage.     

Rapid,gregarious settlement of the larvae of the marine echiuran Urechis caupo Fisher & MacGinitie 1928

Larvae of Urechis caupo Fisher & MacGinitie, reared in the laboratory, were exposed to potential settlement stimuli, including natural sediment from adult burrows, and “scent” obtained from the skin of adult animals. Competent larvae settled rapidly and specifically in response to adult burrow sediment when compared with their responses to other natural and abiotic sediments. Larvae also responded specifically to chemical “scent” from adult animals when the “scent” of another echiuran worm, Listriolobus pelodes Fisher served as a control. Larval responses to chemical “scent” were as great as their responses to natural burrow sediment. Hence, it is likely that larvae settle gregariously in nature in response to “scent” on sediment grains of adult burrows. The chemical “scent” had a molecular weight between ≈3500 and 14000 daltons, as determined by dialysis. It quickly lost its effectiveness in promoting settlement after it was heated to 80 °C, but was relatively stable at ambient ocean temperatures, retaining its effectiveness for several days. It was soluble in sea water. However, larvae did not respond to the chemical “scent”, unless it was adsorbed onto a surface. Purely tactile stimuli, such as the shape, texture, and size-distribution of particles, were not important settlement cues during these experiments.     

Fatty acid composition of the lipids of Puffinus tenuirostris (Temminck) in relation to its diet

The fatty acid composition of lipids isolated from the depot fat, stomach contents, and proventricular oil of adult and chick Puffinus tenuirostris (Temminck) has been analysed. The diet of both adults and chicks is almost exclusively derived from the euphausiid Nyctiphanes australis Sars, and an attempt was made to determine whether dietary lipid affects the composition of depot fat, and whether individual fatty acids in the stomachs and proventricular oil can be used as markers for the origin of the diet. An apparent selectivity in the deposition of fatty acids in the fat depots can be explained by the conversion of fatty alcohols, derived from the euphausiid wax ester, into fatty acids of equivalent chain length and unsaturation. Hexadecadienoic acid appeared to be the only possible marker fatty acid from the euphausiid, but wide variations in its level limits its usefulness as a reliable index of the diet of Puffinus tenuirostris.