Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster

We have characterized biochemical effects of Idh ^GB1 in Drosophila melanogaster. This is a null-activity allele for NADP⁺-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The K _m values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP⁺ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.Research supported by an Alberta Heritage Foundation for Medical Research Establishment Grant to MMB and a Natural Sciences and Engineering Research Council Operating Grant to JHW.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation

Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP⁺-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD⁺-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.Non-standard abbreviations AADC acetoacetate decarboxylase - AcaDH acetaldehyde dehydrogenase - BuaDH butyraldehyde dehydrogenase - CoA-TF acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase - NAD-ADH, NAD⁺ dependent alcohol dehydrogenase - NADP-ADH, NADP⁺ dependent alcohol dehydrogenase     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)     

cell clones and pattern formation: Studies onsevenless,a mutant ofDrosophila melanogaster

Summary sev ^LY3,the only existing allele at thesev locus (1–33,2±0,2), behaves as strongly hypomorph or even as amorph. Ommatidia in asev compound eye have only seven receptor cells, the position of the R7 pattern element being vacant. Various criteria showing that the missing cell is R7 have been verified. These include (i) anatomical characteristics ofsev ommatidia; (ii) behaviour of central R cells insev rdgB double mutants; (iii) medullary projection of central R cell axons; and (iv) mitotic pattern ofsev imaginal discs. The analysis of morphogeneticsev-sev ⁺ mosaics has shown thatsev is expressed autonomously by R7 cells, indicating that thesev phenotype is not due to asev genotype of ommatidial pattern elements other than R7. The study of third instarsev imaginal discs has not brought any direct evidence for death of clustered presumptive R7 cells; however, clonal analysis of the developingsev compound eye has given evidence of developmental parameters comparable to those ofsev ⁺, therefore favouring the hypothesis that R7 cells die insev mutants. On the other hand,sev ⁺ seems to be required for the determination of the R7 cells, since thesev phenotype cannot be uncovered during the last mitoses of heterozygous mutant cells.     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

NADP+ glyceraldehyde-3-phosphate dehydrogenase in soybeans [Glycine max (L.) Merr.]: genetics and developmental expression

Summary Chloroplastic (NADP⁺) glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.9) catalyzes the second reaction in photosynthesis after the fixation of carbon by RuBisCO. Chloroplast-bound (NADP⁺) G3PDH was resolved in soybean by starch gel electrophoresis using l-histidine-citrate buffer (pH 5.7). Histochemical staining revealed zymogram patterns indicative of a tetramer. A survey of soybean genotypes revealed differences in zymogram patterns between the principal cytoplasmic sources of the northern and southern US germplasms. In the soybean pedigree, an allelic frequency shift toward a five-banded pattern was observed. G3PDH polymorphism was due to allele associated with gene expression at the slow locus. No linkage was found between the slow locus of (NADP⁺) G3PDH and AC02, AC03, AC04, ACP, DIA1, IDH1, IDH2, PGM1, and PGM3. Developmental expression in the above-ground tissues was identical, whereas roots as a rule did not express (NADP⁺) G3PDH activity. The importance of chloroplast-bound (NADP⁺) G3PDH in photo-synthesis and its interesting mode of inheritance warrants further exploration of this enzyme in soybean.Technical contribution no. 3293 of the South Carolina Agricultural Experiment Station, Clemson University     

Mechanism of suppression inDrosophila: Regulation of tryptophan oxygenase by thesu(s) + allele

The suppressor gene,su(s)², inDrosophila melanogaster restores the production of red and brown eye pigments for some purple and vermilion mutant alleles, respectively. We showed previously that the product of thesu(s)⁺ allele caused inhibition of the sepiapterin synthase A produced by the purple mutant but did not affect the wild-type enzyme. Suppression was accomplished by removingsu(s)⁺ from the genome. We now report that the tryptophan oxygenase, produced by suppressible vermilion alleles, is also inhibited by extracts fromsu(s)⁺ flies. The inhibition of the vermilion enzyme can be reduced or eliminated, respectively, by prior storage of the extract at 4 or –20°C or by boiling, whereas the wild-type enzyme is not affected by extracts ofsu(s)⁺ flies. Also, when the suppressible vermilion strain is raised on certain diets, brown eye pigment production occurs. This epigenetic suppression was reduced by the presence of an extra copy ofsu(s)⁺ in the genome. These data support a posttranslational mechanism for regulation of enzyme activity in which the activity of the mutant enzyme is reduced by the product of thesu(s)⁺ allele. How thesu(s)⁺ gene product can distinguish between the normal and the mutant forms of these two enzymes is discussed, along with other mechanisms for suppression that are currently under investigation.This work was supported in part by a grant from the KOSEF, Korea Science and Engineering Foundation, and the National Science Foundation under the U.S.-East Asia Cooperative Science Program as well as the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.     

Isolation of a new paramutagenic allele of thesulfurea locus in the tomato cultivar Moneymaker following in vitro culture

A new allele, SC148, of thesulfurea locus inLycopersicon esculentum was detected in a line derived after repeated selfing of plants that had been regenerated from tissue culture. Like the originalsulf mutant, SC148 displayed two mutant phenotypes: green-yellow speckled plants in which thesulf ^vag allele is present and pure yellow plants homozygous for thesulf ^tpura allele. Although the mutant alleles are recessive to wild-type, an unpredictable number of variegated and pura plants appeared in F₁ progenies that had been derived from crosses between SC148 and wild-type tomato plants. The presence of the wild-typesulf ⁺ allele in these variegated heterozygotes was demonstrated using a cytological marker that is linked tosulf. It is concluded that the mutantsulf allele of SC148, imposes its variegated expression state on the wild-typesulf ⁺ allele present insulf ⁺/sulf^vag heterozygotes. This behaviour, known as paramutation, has also been described for the originalsulf allele. The SC148 allele, however, seems to induce changes at an earlier stage in development. The analogy of this paramutagenic system to dominant position effect variegation inDrosophila is discussed.     

Purification,properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B

Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD⁺-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of >1,092 molxmin^-1xmg protein^-1. The enzyme is a hexamer of a polypeptide of Mr=42,500, and the native molecular weight is 250,800. The apparent K _m values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD⁺ in the deamination reaction, and 7.2 mM for -ketoglutarate, 243 mM for NH ₄ ⁺ and 0.028 mM for NADH in the reverse reaction. NADP⁺ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate -ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.     

A calcium-dependent potassium current is increased by a single-gene mutation inParamecium

Summary The membrane currents of wild typeParamecium tetraurelia and the behavioral mutantteaA were analyzed under voltage clamp. TheteaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs⁺ and external TEA⁺. This, along with previous work (Satow, Y., Kung, C., 1976,J. Exp. Biol. 65:51–63) identified this as a K⁺ current. It was further found to be a calcium-activated K⁺ current since this increased outward K⁺ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutationpwB, which blocks the inward calcium current, also blocks this increased outward K⁺ current inteaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K⁺ current are the same for mutant and wild type, theteaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of theteaA mutant. TheteaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of theteaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K⁺ current which prematurely repolarizes the action potential.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Emergence of a mutagenic ochre suppressor mutation under lactose selection in appm mutant ofEscherichia coli harbouring the F′lacZU118 episome

WhenEscherichia coli harbouring theppm (earlier calledadi) mutation and the F′lacZU118 episome is subjected to lactose selection in the presence of suboptimal concentrations of glycerol, Lac⁺ colonies emerge after 5–6 days. They are shown to harbour an ochre suppressor mutation at 15.15 min. Inactivation ofrecA results in approximately four-fold reduction in the response. In theppm — ochre suppressor double mutant background the leakiness of thelacZ allele carried by F′ CC105 is enhanced, suggesting misreading of a valine codon (GUG) as glutamic acid codon (GAG). This is accompanied by reversion of thelacZ mutation tolacZ ⁺ (GTG → GAG). In LB medium both the leakiness and reversion are inhibited by streptomycin. Inactivation ofrecA did not affect leakiness but abolished reversion. These data are discussed in relation to the importance of allele leakiness and restricted growth in stationary-phase (adaptive) mutagenesis.