Effects of waterlogging at different growth stages on the photosynthetic characteristics and grain yield of sorghum (Sorghum bicolor L.).

Various plants, including sorghum (Sorghum bicolor L.), are exposed to waterlogging; however, little is known about the effects of waterlogging at different growth stages on sorghum. A pot experiment was conducted using two sorghum hybrids, Jinuoliang 01 (JN01) and Jinza 31 (JZ31), to investigate the effects of waterlogging at different growth stages on the photosynthesis enzyme activity, chlorophyll content, malondialdehyde (MDA) content, photosynthetic parameters, dry matter accumulation, and grain yield. The experiment was conducted using waterlogging treatments implemented at the five-leaf stage (T1), flowering stage (T2), and filling stage (T3), using standard management (no waterlogging) as a control (CK). The adverse effects of waterlogging on sorghum growth varied with the waterlogging timing, with the maximum impact at T1, followed by T2 and T3. JZ31 was more sensitive to waterlogging compared to JN01. Waterlogged conditions inhibited the photosynthetic enzyme activity and reduced the chlorophyll content and photosynthesis, ultimately lowering the biomass yield and grain yield. The maximum yield loss was observed with the T1 waterlogging treatment; the grain yield of JN01 and JZ31 decreased by 52.01-54.58% and 69.52-71.97%, respectively, compared with CK. Furthermore, the decline in grain yield in T1 was associated with reducing grain number per panicle. These findings indicate that sorghum is sensitive to waterlogging at the five-leaf stage and JZ31 is more sensitive to waterlogging than JN01, which may provide a basis for selecting genotypes and management measures to cope with waterlogging in sorghum.

Characterisation of seeds of a C4 phosphoenolpyruvate carboxylase-deficient mutant of Amaranthus edulis.

Seeds from the C(4) plant Amaranthus edulis were studied as part of the characterisation of a mutant (designated LaC(4) 2.16), which contains reduced amounts (5% of wild type) of the photosynthetic leaf form of phosphoenolpyruvate carboxylase (PEPC). On a per seed basis, the amount of PEPC activity was not significantly altered, while the weight and protein content of the mutant seeds were 34% lower than that of the wild type. Western gel blot analysis detected two PEPC polypeptides with molecular masses of 105 kDa (minor) and 100 kDa (major). The determination of in vitro phosphorylation in reconstituted assays revealed the presence of both calcium-dependent and calcium-independent PEPC-kinase activities in protein extracts of wild-type and mutant seeds. However, PEPC proteins were phosphorylated in dry seeds, and PEPC phosphorylation did not occur in vivo during seed imbibition in the presence of (32) P-phosphate. In contrast, (32) P-phosphate was incorporated into a range of proteins in wild-type seeds, but not in mutant seeds. In addition, ATP content was much reduced in germinating mutant seeds and this did not increase following the supply of phosphate. Collectively, these data suggest that the deficiency in C(4) PEPC in mutant A. edulis leaves has no effect on C(3) -type PEPC content and phosphorylation state in seeds, but causes impairment of energy production, thereby accounting for the reduced germination of the mutant.

Diversity in forms of C4 in the genus Cleome (Cleomaceae).

BACKGROUND AND AIMS: Cleomaceae is one of 19 angiosperm families in which C(4) photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C(4) in genus Cleome. Methods Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC). KEY RESULTS: Three species with C(4)-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C(4) photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C(4) cycle (compared with the C(3)-C(4) intermediate C. paradoxa and the C(3) species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells. CONCLUSIONS: NAD-malic enzyme-type C(4) photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C(3)-C(4) intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.

Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032.

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.

Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes.

Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).

The effect of NADP-dependent malic enzyme expression and anaerobic C4 metabolism in Escherichia coli compared with other anaplerotic enzymes.

AIMS: To understand the modification of C4-metabolism under anaerobic glycolysis condition by overexpressing anaplerotic enzymes, which mediating carboxylation of C3 into C4 metabolites, in Escherichia coli. METHODS AND RESULTS: Anaplerotic NADP-dependent malic enzyme (MaeB), as well as the other anaplerotic enzymes, including phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate carboxykinase (Pck) and NAD-dependent malic enzyme (MaeA), were artificially expressed and their C4 metabolism was compared in E. coli. Increasing MaeB expression enhanced the production of C4 metabolites by 2.4 times compared to the wild-type strain in anaerobic glucose medium with bicarbonate supplementation. In MaeB expression, C4 metabolism by supplementing 10 g l(-1) of NaHCO(3) was three times than that by no supplementation, which showed the greatest response to increased CO(2) availability among the tested anaplerotic enzyme expressions. CONCLUSIONS: The higher C4 metabolism was achieved in E. coli expressing increased levels of the NADPH-dependent MaeB. The greatest increase in the C4 metabolite ratio compared to the other tested enzymes were also found in E. coli with enhanced MaeB expression as CO(2) availability increased. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher C4 metabolites and related biomolecule productions can be accomplished by MaeB overexpression in metabolically engineered E. coli.

Effects of dexamethasone on the glucogenic capacity of fetal, pregnant, and non-pregnant adult sheep.

Fetal glucocorticoids have an important role in the pre-partum maturation of physiological systems essential for neonatal survival such as glucogenesis. Consequently, in clinical practice, synthetic glucocorticoids, like dexamethasone, are given routinely to pregnant women threatened with pre-term delivery to improve the viability of their infants. However, little is known about the effects of maternal dexamethasone treatment on the glucogenic capacity of either the fetus or mother. This study investigated the effects of dexamethasone treatment using a clinically relevant dose and regime on glycogen deposition and the activities of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the liver and kidney of pregnant ewes and their fetuses, and of non-pregnant ewes. Dexamethasone administration increased the glycogen content of both the fetal and adult liver within 36 h of beginning treatment. It also increased G6Pase activity in the liver and kidney of the fetuses but not of their mothers or the non-pregnant ewes. Neither hepatic nor renal PEPCK activity was affected by dexamethasone in any group of animals. These changes in glycogen content and G6Pase activity were accompanied by rises in the plasma glucose and insulin concentrations and by a fall in the plasma cortisol level in the fetus and both groups of adult animals. In addition, dexamethasone treatment raised fetal plasma tri-iodothyronine (T(3)) concentrations and reduced maternal levels of plasma T(3) and thyroxine, but had no effect on thyroid hormone concentrations in the non-pregnant ewes. These findings show that maternal dexamethasone treatment increases the glucogenic capacity of both the mother and fetus and has major implications for glucose availability both before and after birth.

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta.

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades--namely, the leaf sheath, stem, scale leaf, and constituents of the spike--also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

Immunolocalization of carbonic anhydrase and phosphoenolpyruvate carboxylase in developing seeds of Medicago sativa.

To investigate the role of carbonic anhydrase (CA; EC 4.2.1.1) and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) during Medicago sativa seed development, the distribution of both proteins was examined using an immunohistological approach. Both enzymes are co-localized in most ovular and embryonic tissues. In early stages of seed development, both proteins were abundant in embryo and integuments, while at subsequent stages both proteins are accumulated in endosperm, nucellus and integuments. At late stages of seed development when both endosperm and nucellus are degraded, significant accumulation of both proteins was observed in the embryo proper. Chlorophyll was found to accumulate in embryos after the heart stage and reached a maximum at mature stage. It is suggested that CA and PEPC play a role in respiratory carbon dioxide refixation while generating malate to support amino acid and/or fatty acids biosynthesis.

A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: application to the analysis of several glycolytic enzymes.

We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and pyruvate kinase from plant tissues. It can be applied to the detection of all enzymes which are normally detected spectrophotometrically using coupled enzyme assays consuming NAD(P)H.

[Specific density of leaf as a characteristic of the photosynthetic apparatus]. / Udel'naia plotnost' lista kak kharakteristika fotosinteticheskogo apparata.

At early stages of ontogeny (up to 50-60% of the maximum leaf area) of wheat (Triticum aestivum L.), meadow fescue (Festuca pratensis Huds.), reed fescue (F. arindinacea Schreb.), and sugar beet (Beta vulgaris L. var. saccharifera (Alef) Krass), there is correlation between changes in the specific leaf density (SLD), rate of photosynthetic CO2 assimilation; activity of the key photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39); and concentration of chlorophyll (Chl) a, Chl b, carotenoids, and soluble leaf proteins. However, there is no correlation of SLD with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). Senescence of leaves was accompanied by a decrease in the SLD value. Treatment with cytokininomimetics (6-benzylaminopurine and Metribuzin) caused an increase in the SLD value. The specific leaf density is suggested to be a structural and functional characteristic of the photosynthetic apparatus of agricultural plants.

Phosphorus deficiency in Lupinus albus. Altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase.

The development of clustered tertiary lateral roots (proteoid roots) and the expression of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in roots were studied in white lupin (Lupinus albus L.) grown with either 1 mM P (+P-treated) or without P (-P-treated). The +P-treated plants initiated fewer clustered tertiary meristems and the emergence of these meristems was delayed compared with - P-treated plants. Proteoid root zones could be identified 9 d after emergence in both P treatments. Amounts of PEPC mRNA, PEPC specific activity, and enzyme protein were greater in proteoid roots than in normal roots beginning at 10, 12, and 14 d after emergence, respectively. The increases in PEPC mRNA, PEPC enzyme, and PEPC specific activity suggest that this enzyme is in part under transcriptional regulation. Recovery of organic acids from root exudates coincided with the increases in PEPC specific activity. The -P-treated plants exuded 40-, 20-, and 5-fold more citrate, malate, and succinate, respectively, than did +P-treated plants. Data presented support the hypothesis that white lupin has concerted regulation of proteoid root development, transcriptional regulation of PEPC, and biosynthesis of organic acids for exudation in response to P deficiency.

A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacterium Synechocystis PCC6803.

The involvement of a gene of Synechocystis PCC6803, icfG, in the co-ordinated regulation of inorganic carbon and glucose metabolism, was established. The icfG gene codes for a 72 kDa protein, which shows no homology with those registered in data libraries. Expression of icfG required glucose, the actual inducer probably being glucose-6-phosphate, and was independent of light and of the external inorganic carbon concentration. Mutants carrying an inactivated copy of icfG were constructed. Their growth characteristics were identical to those of the wild type under all regimes except in limiting inorganic carbon with glucose being present either before or after the transfer to the limiting conditions. These conditions completely prevented growth, both in the light and in the dark. The inhibition could be relieved by several intermediates of the tricarboxylic acid cycle. Assays of various enzymic activities related to inorganic carbon uptake and to its assimilation via either the Calvin cycle or phosphoenolpyruvate carboxylase did not reveal the level of action of IcfG. Possible models include a blockage of the assimilation of both carbon sources in the absence of IcfG, or the inhibition of Ci incorporation route(s) essential under limiting inorganic carbon conditions, even when glucose is present, and even in the dark.

Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase.

To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented.

Enzymatic determination of total CO2 in freeze-clamped animal tissues and plasma.

An enzymatic method for measuring total carbon dioxide content in freeze-clamped animal tissues is described. Total carbon dioxide content [TCO2] was defined as the sum of the dissolved CO2, the bicarbonate concentration, and the carbonate concentration. Tissue was extracted in 80% methanol, 20 mM 2-amino-2-methyl-1-propanol, pH 9.5 at 25 degrees C and homogenized in a 1.5-ml Sardstat screw-top test tube containing 0.5-mm glass beads and a minibead beater. Total CO2 was determined as bicarbonate/carbonate by monitoring the oxidation of NADH at 340 nm using the coupled assay of phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). In the coupled assay system, 1 mumol of bicarbonate/carbonate consumed is equivalent to the oxidation of 1 mumol NADH at 340 nm. The assay medium comprised 50 mM 2-amino-2-methyl-1-propanol, pH 9.0 at 25 degrees C, 5 mM phosphoenolpyruvate (PEP), 0.25 mM NADH, 5 mM MgCl2, 5 mM mercaptoethanol, 0.02% bovine serum albumin, 10 mM oxamate, PEP carboxylase (0.5 units/ml), and malate dehydrogenase (0.5 units/ml). The total CO2 content measured in freeze-clamped rat heart, liver, brain, and skeletal muscle was 20.53 +/- 0.64, 17.34 +/- 0.67, 17.00 +/- 0.48, 16.06 +/- 0.53 mumol/g wet wt tissue, respectively (n = 5). The total CO2 in the crusher muscle of the lobster was found to be 5.0 +/- 0.33 mumol/g wet wt. Total CO2 was also enzymatically measured in arterial plasma from four chronically cannulated male wistar rats and was 24.65 +/- 1.81 mumol/ml plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Key enzymes of gluconeogenesis are dose-dependently reduced in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats.

Male Sprague-Dawley rats (240-245 g) were dosed ip with 5, 15, 25, or 125 micrograms/kg -,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. Ad libitum-fed and pair-fed controls received vehicle (4 ml/kg) alone. Two or 8 days after dosing five rats of each group were sacrificed, their livers removed and assayed for the activities of three gluconeogenic enzymes [phosphoenol-pyruvate carboxykinase (PEPCK; EC 4.1.1.32), pyruvate carboxylase (PC; EC 6.4.1.1), and glucose-6-phosphatase (G-6-Pase, EC 3.13.9)], and one glycolytic enzyme [pyruvate kinase (PK; EC 2.7.1.40)] by established procedures. The activity of PK was not affected by TCDD at either time point. The activity of G-6-Pase tended to be decreased in TCDD-treated animals, as compared to pair-fed controls, but the decrease was variable without an apparent dose-response. The activity of PEPCK was significantly decreased 2 days after dosing, but a clear dose-response was apparent only at the 8-day time point. Maximum loss of activity at the highest dose was 56% below pair-fed control levels. PC activity was slightly decreased 2 days after TCDD treatment and displayed statistically significant, dose-dependent reduction by 8 days after dosing with a 49% loss of enzyme activity after the highest dose. It is concluded that inhibition of gluconeogenesis by TCDD previously demonstrated in vivo is probably due to decreased activities of PEPCK and PC. The data also support the prevailing view that PEPCK and PC are rate-determining enzymes in gluconeogenesis.

Regulation of the aggregation state of maize phosphoenolpyruvate carboxylase: evidence from dynamic light-scattering measurements.

The molecular weights of different aggregational states of phosphoenolpyruvate carboxylase purified from the leaves of Zea mays have been determined by measurement of the molecular diameter using a Malvern dynamic light scattering spectrometer. Using these data to identify the monomer, dimer, tetramer, and larger aggregate(s) the effect of pH and various ligands on the aggregational equilibria of this enzyme have been determined. At neutral pH the enzyme favored the tetrameric form. At both low and high pH the tetramer dissociated, followed by aggregation to a "large" inactive form. The order of dissociation at least at low pH appeared to be two-step: from tetramer to dimers followed by dimer to monomers. The monomers then aggregate to a large aggregate, which is inactive. The presence of EDTA at pH 8 protected the enzyme against both inactivation and large aggregate formation. Dilution of the enzyme at pH 7 at room temperature results in driving the equilibrium from tetramer to dimer. The presence of malate with EDTA stabilizes the dimer as the predominant form at low protein concentrations. The presence of the substrate phosphoenolpyruvate alone and with magnesium and bicarbonate induced formation of the tetramer, and decreased the dissociation constant (Kd) of the tetrameric form. The inhibitor malate, however, induced dissociation of the tetramer as evidenced by an increase in the Kd of the tetramer.

A microtiter plate-based assay for phosphoenolpyruvate carboxylase.

A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography.

Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Identification of an amino acid substitution in the mutant.

The structural gene encoding a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate (Fru-P2) was isolated from total E. coli PpcI genomic DNA. This mutant gene is located on a 4.4-kilobase SalI DNA fragment which, when ligated to SalI-digested pBR322, resulted in the generation of the plasmid pFS16. Detailed restriction mapping of the wild-type and mutant genes for phosphoenolpyruvate carboxylase revealed the presence of a ClaI restriction site at position 563 of the mutant gene only. This ClaI site is located on a 289 PvuII/DdeI fragment which codes for amino acid residues 174-270 of the phosphoenolpyruvate carboxylase enzyme. When this portion of the mutant gene is present in chimeras of the wild-type and mutant genes, the phosphoenolpyruvate carboxylase produced cannot be activated by Fru-P2. The mutation resulting in the generation of the ClaI site in the mutant gene has also resulted in an amino acid substitution at residue 188; threonine in the wild-type enzyme has been replaced by isoleucine in the mutant enzyme. Comparison of the nucleotide sequence of this 289-base pair PvuII/DdeI region of the mutant gene with its homologous region in the wild-type gene verified that this mutation, which resulted in the generation of the ClaI site, is the only change that has occurred on this 289-base pair fragment of the mutant gene, and thus the amino acid replacement of threonine by isoleucine is the only change that could be linked to the inability of the mutant enzyme to be activated by Fru-P2.