Characterization of a light-dependent glutamate synthase activity in Chlamydomonas reinhardtii

Photosynthetically active vesicles prepared from Chlamydomonas reinhardtii retained a light-dependent glutamate synthase activity which was highly specific for 2-oxoglutarate (K_m=2.1 mM) and L-glutamine (K_m=0.9 mM) as amido group acceptor and donor respectively. This activity was inhibited by azaserine, p-hydroxymercuribenzoate and 3-(p-chlorophenyl)-1,1-dimethyl urea.Light-dependent synthesis of glutamate was also obtained by coupling Chlamydomonas photosynthetic particles to purified ferredoxin-glutamate synthase, using ascorbate and 2,6-dichlorophenol-indophenol as electron donor. This system was also specific for 2-oxoglutarate (K_m=1 mM) and L-glutamine (K_m=0.8 mM) as substrates, and was stimulated by dithioerythritol. Azaserine and p-hydroxymercuribenzoate, but not 3-(p-chlorophenyl)-1,1-dimethyl urea, inhibited the reconstituted activity; high concentrations of 2-oxoglutarate were inhibitory.Abbreviations A Absorbance - CCP p-Trichlorometoxi-carbonylcyanide-phenylhydrazone - Chl Chlorophyll - CMU 3-(p-Chlorophenyl)-1,1-dimethyl urea - DPIP 2,6-Dichlorophenol-indophenol - DTE Dithioerythritol - MSX L-Methionine, D-L, sulfoximine - MV Methyl viologen     

Selective emergence and survival of early polypeptides in water

Oligopeptides essential to primitive cells could not be obtained just by raising the background noise of organic compounds produced by a prebiotic chemistry working at random. Selection pathways were required. Experimental evidence is given for selective condensation of amino acids in water as well as for selective resistance to degradation. It is shown that N-carboxyanhydrides are good candidates for chemical selection in water. They are formed when active esters of amino acids are left in the presence of bicarbonate ions or when N,N'-carbonyldiimidazole is used as condensing agent. Polymerization of a mixture of proteinaceous and non-proteinaceous amino acids leads to an enrichment in the proteinaceous ones plusα-aminobutyric acid. Selective resistance toward degradation ofβ-pleated sheet conformation is used to exemplify a possible accumulation of homochiral sequences made of hydrophilic and strong hydrophobic residues. Amino acids with branched aliphatic side-chains are selected but those having short linear aliphatic side-chains such asα-aminobutyric acid or norvaline are not.     

Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX)

Summary The changes in cell wall strength of Hansenula polymorpha have been investigated in continuous cultures with respect to the recovery of methanol oxidase (MOX). Cultures grown on several substrate mixtures that enable induction of MOX have been compared with cultures grown on methanol as the sole inducer. The effects of dilution rate (D) on lysis properties have been studied. The cell wall strength was consistently influenced by growth media and D. Media containing glycerol/methanol showed the slowest lysis kinetics, with a large fraction of non-degradable cell wall material. In continuous cultures grown on a mixture of glucose and methanol both the resistance to zymolyase and the mean cell wall thickness increased at D<0.1 h^–1. The yield of MOX by zymolyase lysis is reproducible and up to 100% higher than that of the standard ultrasonic treatment. The lysis kinetics indicated that zymolyase punctures the cell wall; since the release rate of MOX is lower than that of protein, the cell contents will leak through. At D-values>0.2 h^–1, both protein and MOX release rates increase, reflecting a change in lysis mechanism due to the increased fraction of thin daughter cells. Kinetic analysis of zymolyase lysis using both physical and enzymatic methods provides information for achieving optimal recovery of MOX.Abbreviations and symbols C _MOX MOX activity [MOX units·g X^–1] - D dilution rate [h^–1] - MOX methanol oxidase - k_c decay rate constant of A 610 nm [min^–1] - k_d decay constant of MOX activity [min^–1] - k_dis dissociation rate constant [min^–1] - k_MOX release rate constant of MOX activity [min^–1] - k_p release rate constant of protein [min^–1] - R recovery efficiency of enzyme [-] - St stability of enzyme [-]     

Regulation of the amylase secretion by the yeast Filobasidium capsuligenum and a 2-deoxy-d-glucose resistant mutant

Summary The regulation of extracellular amylase production by the basidiomycetous yeast Filobasidium capsuligenum CCY 64-5-1 was characterized using growing and resting cells. A basal level of amylolytic activity was produced with various carbon sources including glucose. Amylase secretion was repressed by glucose and, more severely, by 2-deoxy-d-glucose, whereas compounds with -1,4-linked glucose, such as methyl glucoside, maltose, -cyclodextrin and soluble starch, served as inducers. Repression was not relieved by exogenously added cAMP. The effects of several metabolic inhibitors on amylase secretion were studied. Following UV-mutagenesis a mutant strain (FC-5) capable of growing in a 2-deoxy-d-glucose supplemented corn starch medium was selected for further characterization. This strain produced more amylase, had acquired an increased resistance against repression by glucose, and retained a growth rate comparable to the wild type. FC-5 was also characterized by a reduced glucokinase activity and an increased hexokinase activity.     

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.     

Insulin and IGF receptors are developmentally regulated in the chick embryo eye lens

We have previously reported that insulin-like growth factor (IGF) receptors appear to predominate over insulin receptors in early stages of embryogenesis in the chick (days 2-3 whole embryo membranes). Overall, [125I]IGF I and II binding to specific receptors was maximal when the rate of brain growth is highest. In the present study we used the embryonic chick lens, a well-defined tissue composed of a single type of cell, to analyse whether changes of insulin and IGF I binding are correlated with changes in growth rate and differentiation state of the cells. We show that both insulin receptors and IGF receptors are present in the lens epithelial cells, and that each type is distinctly regulated throughout development. While there is a direct correlation between IGF-binding capability and growth rate of the cells, there is less relation to differentiation status and embryo age. Insulin receptors, by contrast, appear to be mostly related to the differentiated state of cells, decreasing sharply in fibers, irrespective of their developmental age.     

Possible role of cytokinins in cereals with regard to the resistance to obligate fungus parasites

The cultivars of barley and wheat resistant to mildew had a higher level of free zeatin and its derivatives during the whole ontogeny than the susceptible cultivars. The effect of exogenous application of cytokinins on the growth ofErysiphe graminis DC. was different. Kinetin and benzylaminopurine showed only a slight inhibitory effect. Zeatin and its derivatives completely inhibited growth of this fungus. Presented at the International Symposium “Plant Growth Regulators” held on June 18-22 1984 at Liblice, Czechoslovakia.     

Presence of nucleosomes inPenicillium chrysogenum

We have studied the chromatin structure ofPenicillium chrysogenum. This fungus presents the typical nucleosomal repeat and the core DNA size characteristic of all the eukaryotes. The repeat length (about 180 base pairs) is in the range of those obtained for most fungi (160–180 base pairs) and shorter than in higher eukaryotes. Knowledge aboutP. chrysogenum chromatin structure opens the way to the study of the mechanisms of genetic regulation in this filamentous fungus.     

A complex balanced chromosomal rearrangement in repeated abortions

Summary A double balanced reciprocal translocation involving four chromosomes, t(1;19;6;14) (1p11; 19p11; 6q25; 14q21), was found in the phenotypically normal husband in a couple referred because of repeated abortions. Reciprocal translocations, t(6;14), had been transmitted by his mother, his father being apparently homozygous for a translocation comprising pairs 1 and 19-t(1;19)(1;19). The genetic consequences of this complex chromosomal rearrangement are analyzed.