Effects of supplementing a CP-reduced diet with rumen-protected methionine on Fleckvieh bull fattening

The objective of this study was to evaluate the effect of supplementing a CP-reduced diet with rumen-protected methionine on growth performance of Fleckvieh bulls. A total of 69 bulls (367 ± 25 kg BW) were assigned to three feeding groups (n = 23 per group). The control (CON) diet contained 13.7% CP and 2.11 g methionine/kg diet (both DM basis) and was set as positive control. The diet reduced in CP (nitrogen) (RED) diet as negative control and the experimental RED + rumen-protected methionine (MET) diet were characterised by deficient CP concentrations (both 9.04% CP). The RED + MET diet differed from the RED diet in methionine concentration (2.54 g/kg DM vs. 1.56 g/kg DM, respectively) due to supplementation of rumen-protected methionine. Rumen-protected lysine was added to both RED and RED + MET at 2.7 g/kg DM to ensure a sufficient lysine supply relative to total and metabolisable protein intake. Metabolisable energy (ME) and nutrient composition were similar for CON, RED, and RED + MET. Bulls were fed for 105 days (d) on average. Individual feed intake was recorded daily; individual BW was recorded at the beginning of the experiment, once per month, and directly before slaughter. At slaughter, blood samples were collected and carcass traits were assessed. Reduction in dietary CP concentration reduced feed intake, and in combination with lower dietary CP concentration, daily intake of CP for RED and RED + MET was lower compared with CON (P < 0.01). Daily ME intake was reduced in RED and RED + MET compared with CON (P < 0.01). Consequently growth performance and carcass weights were reduced (both P < 0.01) in both RED and RED + MET compared with CON. Supplemental rumen-protected methionine was reflected in increased serum methionine concentration in RED + MET (P < 0.05) as compared to RED but it did not affect growth performance, carcass traits and serum amino acid (AA) concentrations, except for lysine which was reduced (P < 0.01) compared to CON and RED. In conclusion, bulls fed RED or RED + MET diets were exposed to a ruminal CP deficit and subsequently a deficit of prececal digestible protein, but methionine did not appear to be the first-limiting essential AA for growth under the respective experimental conditions.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。     

MTT比色法检测赖氨酸、蛋氨酸对体外培养的奶牛乳腺上皮细胞增殖的影响

采用噻唑蓝比色法检测赖氨酸、蛋氨酸对体外培养的奶牛乳腺上皮细胞增殖的影响。赖氨酸和蛋氨酸在培养基中的添加浓度分别为0、0.05、0.2、0.4、0.8、1.6、3.2、6.4、12.8、25.6mmol/L和0、0.025、0.1、0.2、0.4、0.8、1.6、3.2、6.4、12.8mmol/L;培养期为24、48和72h。结果表明,赖氨酸在0.8-1.6mmol/L、蛋氨酸在0.4-0.8mmol/L浓度范围内对体外培养的奶牛乳腺上皮细胞增殖的促进作用最明显且在48h时增殖作用最强(P0.0001)。     

Effect of dimethyl sulfoxide on lysine production by a mutant ofBacillus subtilis with low homoserine dehydrogenase activity

SomeBacillus subtilis mutants with different levels of homoserine dehydrogenase were described. Strains that do not accumulate methionine have a high homoserine dehydrogenase activity. Low activity was detected in mutants where cell growth was completely inhibited by 0.7 mmol/L methionine. A low concentration of dimethyl sulfoxide had a stimulatory effect on lysine production by the methionine-sensitive mutant ofBacillus subtilis.     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

Control of methionine synthesis by lysine in Lemna minor

When Lemna minor was cultured in the presence of 0.25 mM l-lysine, the concentration of free methionine and formyl and methyl tetrahydrofolate (THFA) were decreased. l-lysine, l-homoserine, l-threonine and l-methionine at concentrations up to 8 mM did not affect N¹⁰-formyl THFA synthetase (E.C. 6.3.4.3) and N⁵,N¹⁰-methylene THFA reductase (E.C. 1.1.1.68). In contrast, serine hydroxymethyltransferase (E.C. 2.1.2.1) activity was inhibited by lysine. This inhibition gave a sigmoidal curve when plotted for a range of l-lysine or THFA concentrations. Exogenous lysine also reduced the incorporation of glycine [¹⁴C] and serine [3-¹⁴C] into free and protein methionine. Lysine, which is known to control synthesis of homocysteine in L. minor, may also regulate production of C-1 units for methionine synthesis by inhibition of serine hydroxymethyltransferase.     

Untersuchungen über den Vitamin B2-Bedarf der Legehenne

Because of the well established function of carnitine possible effects of carnitine were studied in poultry. In trial I it was investigated if carnitine and its precursors (lysine, methionine) reduce the formation of abdominal fat in broilers. Chickens (10 groups of 10 chickens each) were fed different diets (control, lysine and methionine in excess and deficient, respectively, with or without 5% fat supplement, L‐carnitine and DL‐carnitine supplement, respectively).Performance (body weight gain, feed conversion), amount of abdominal fat and carnitine concentration in blood, muscles (M. sartorius, M.pectoralis superficialis, cardiac), liver and kidney were determined. Performance and abdominal fat were influenced by dietary fat, lysine and methionine as expected and were not altered by carnitine. Excess and deficiency of lysine and methionine did not influence, fat supplement reduced and carnitine supplementation significantly increased tissue concentration of carnitine.

In trial II it was studied if supplementation of a commercial layers’ ration with either 500 mg L‐carnitine or 500 mg nicotinic acid or both per kg reduces the cholesterol concentration in yolk. Influence on body weight, feed intake, laying performance, serum and yolk cholesterol concentration could not be observed, but yolk concentration of carnitine was significantly increased in supplemented groups.

Trial III should clarify if the L‐carnitine content in broiler parentstock ration influences hatchability. Four groups of 1350 hens each were fed a commercial all‐mash supplemented with 0, 20, 50 and 100 mg L‐carnitine, respectively. Hatching rate was increased from 83% to 87% and from 82.4% to 85.3% in groups supplemented with 50 and 100 mg L‐carnitine, respectively, and in randomly sampled eggs of these groups carnitine concentration in yolk was higher.     

Characterization of cultured tobacco cell lines resistant to ethionine, a methionine analog

Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110×), threonine (18×), and lysine (5×). In vitro enzymic analysis of lysine-sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K_m values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression.     

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca²⁺]_i) and receptor-activated (carbachol, 0.1 mmol/L) Ca²⁺ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca²⁺]_i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca²⁺]_i plays a significant role for acrylamide-induced axonopathy.     

l-Phenylalanine production by double auxotrophic mutants ofArthrobacter globiformis

A number of tryptophan-plus-tyrosine double auxotrophs have been isolated from a glutamate producingArthrobacter globiformis excretingl-phenylalanine by two-step mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. For the three potent mutants tested the medium of Alföldi was found to be the best. The optimum tryptophan, tyrosine and biotin concentrations for phenylalanine production of these mutants were 0.5 mmol/L, 0.1 mmol/L and 5 μg/L, respectively. At these levels strain TT-39 yielded 2.6 g phenylalanine per L of medium in flask culture with glucose (350 mmol/L) and NH₄Cl (60 mmol/L).     

Lupinus luteus,Vicia sativa and Lathyrus cicera as protein sources for piglets: ileal and total tract apparent digestibility of amino acids and antigenic effects

Twenty-four male piglets, weaned at 28 days of age, were used to measure the total and ileal digestibility and serum immune responses to dietary leguminous seeds. The experimental diets consisted of a control starter (C) and three other diets prepared by replacing 30% of the crude protein content of the C diet by the protein of Lupinus luteus (LL), Vicia sativa (VS) or Lathyrus cicera (LC). The total tract apparent digestibility (TTAD) of energy and crude protein (CP) was lowest (P<0.001) for the LC diet. Similarly TTAD of lysine, methionine and threonine were also lowest for the LC diet. The apparent ileal digestibility (AID) of lysine, methionine and threonine were lowest for the LC diet. AID of lysine and methionine was highest for the LL seeds (0.764 and 0.834, respectively) and lowest (P<0.05) for the LC seeds (only 0.454 and 0.420, respectively). Immunoblots of individual pig serum were made to detect both residual antigenic storage proteins of the seeds of each legume used and IgGs specific to those storage proteins. Antibodies against β-conglutin of L. luteus, vicilin of V. sativa and vicilin of L. cicera were detected 28 days after feeding the diet in the sera of piglets fed on the LL, VS or LC diets, respectively. Conversely, no storage protein was found in the serum of any piglet fed either on LL, VS or LC diets. The presence of antibodies against β-conglutin of lupine, vicilin of V. sativa and vicilin of L. cicera, respectively indicated an immune response in weaned piglets. The absence of residual antigenic proteins may be due to the digestive adaptation of the piglet to the legume-based diets. However, no direct relation between the differences in digestibility coefficients among the legume seeds and their antigenicity was established.     

l-lysine production by S-2-aminoethyl-l-cysteine-resistant mutants ofArthrobacter globiformis

Using mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a number of homoserine auxotrophs have been isolated from a glutamate-producing Arthrobacter globiformis excreting L-lysine in good amounts. For further improvement, mutants resistant to the lysine analog S-(2-aminoethyl)-L-cysteine have been isolated from homoserine auxotrophs. For the three potent mutants tested, White's medium was found to be the best. Glucose, ammonium nitrate and biotin were found to be optimum at 280 mmol/L, 40 mmol/L and 22 nmol/L, respectively. With optimal glucose, ammonium nitrate and biotin, the strain AECrVI yielded 36 g lysine per L in flask culture.     

Determination of nutrient requirements for growth and maintenance of growing pigs under tropical condition

Data from 27 feeding trials conducted on growing pigs from different research institutes across India were subjected to mixed model regression analysis to derive requirements of digestible energy (DE), crude protein (CP) and essential amino acids for maintenance and body weight gains. The ranges of maintenance requirements were determined to be: DE 516 to 702 kJ/kg M^0.75, CP 6.98 to 11.62, lysine 0.431 to 0.664, methionine 0.265 to 0.458, methionine + cystine 0.327 to 0.466, cystine 0.055 to 0.184, threonine 0.205 to 0.511, arginine 0.377 to 1.21, isoleucine 0.241 to 0.775, leucine 0.604 to 1.54, phenylalanine + tyrosine 0.496 to 1.33, tryptophan 0.078 to 0.213, and valine 0.330 to 0.892 g/kg M^0.75, respectively for different body weight ranges. The corresponding requirements for 1 g gain in body weight were: DE 28.6 to 38.6 kJ, CP 0.27 to 0.44 g, lysine 0.0071 to 0.0126 g, methionine 0.0047 to 0.0133 g, methionine + cystine 0.0151 to 0.0261 g, cystine 0.0043 to 0.0094 g, threonine 0.0052 to 0.0165 g, arginine 0.0045 to 0.0301 g, isoleucine 0.0023 to 0.0198 g, leucine 0.0150 to 0.0447 g, phenylalanine + tyrosine 0.0091 to 0.0382 g, tryptophan 0.0005 to 0.0044 g, and valine 0.0061 to 0.0222 g. Regression equations had high R² values (ranging from 0.50 to 0.99 for different estimates), low coefficients of variation, low variance of error estimates and the coefficients were highly significant (P < 0.001). Regressed values were used to develop feeding standards. As the new standards derived in the present study are based on a thorough analysis of a larger database than previous Indian standards, the new feeding standard seems to be more appropriate for India and other tropical countries.     

Selection and Characterization of Asparagus Mutants Resistant to Inhibition by Lysine Plus Threonine

Asparagus officinalis calli were induced from shoot of seedlings. After mutagenization, two lysine plus threonine resistant mutant lines (LTR2, LTR3) were obtained by selectionnonselection-reselection procedures with 2 mmol/1 lysine plus threonine. LTR2 and LTR3 caIli remained resistance to lysine plus threonine after being subcultured for 1 year, and both of them showed cross resistance to 1 mmol/l aminoethylcysteine. In resistant calli, the free lysine, methionine and an unknown amino acid were l-l0 times more than those in controls.     

Cerato-ulmin—a wilting toxin of Dutch elm disease fungus

Cerato-ulmin, a toxin produced by Ceratocystis ulmi, the causal agent of Dutch elm disease, has been characterized as a small protein (128 residues) with a MW of ca 13000. The protein has a high content of cystine, proline, leucine, serine and aspartic acid/asparagine; it is low in histidine, lysine, arginine, isoleucine, phenylalanine and tyrosine and does not contain cysteine, methionine, or tryptophan. The amino acid sequence of the N-terminal region is: H₂N-Ala-Asp-Ser-Tyr-Asp-Pro-Cys-Thr-Gly-Leu-Leu-Gln-Lys-Ser-Pro-Gln-Cys-Cys-Asp-Thr-Asp-Ile-Leu-Gly-Val-Ser-Asp-Leu-Asp-Cys-. Toxic symptoms similar to those of Dutch elm disease can be elicited by cerato-ulmin in white elm shoot cuttings (Ulmus americana L.).     

Identification and characterization of an anti-fungi Fusarium oxysporum f. sp. cucumerium protease from the Bacillus subtilis strain N7

A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30–60% (NH₄)₂SO₄ precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOF? Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two β-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 μg/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0–10.6 and 70°C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na⁺, Fe³⁺, Cu²⁺, Mg²⁺ (5 mmol/L each) and H₂O₂ (2%, v/v). However, protease activity was activated by Ca²⁺, K⁺, Mn²⁺ (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In additon, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).     

Feedback-insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine

The regulatory properties of aspartate kinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) in two barley (Hordeum vulgare L.) mutants resistant to growth inhibition by lysine plus threonine, Rothamsted (R) 3004 and R3202, were compared with those in the normal, sensitive parent line cv. Bomi. Three forms of aspartate kinase (AKI, AKII, AKIII) were chromatographically separated and were considered to represent at least three independently regulated isoenzymes. Aspartate kinase I was inhibited by threonine; AKII and AKIII by lysine or lysine plus S-adenosylmethionine. The characteristics of AKI were unchanged in the mutants. Aspartate kinase II and AKIII from Bomi were both inhibited by lysine and by lysine plus S-adenosylmethionine. Aspartate kinase II from mutant R3202 was altered in its properties such that it was insensitive to lysine or lysine plus S-adenosylmethionine; AKII from mutant R3004 did not differ in its properties from AKII of Bomi. The concentration of lysine required to give half maximal inhibition of AKIII from R3004 was ten times that required for AKIII of Bomi; AKIII from R3202 did not differ from that of Bomi in this regard. There was no change in the properties of homoserine dehydrogenase of the mutants as compared with that of Bomi. We conclude that the lt1 and lt2 loci code for structural genes for lysine- and lysine plus S-adenosylmethionine-sensitive aspartate kinase isoenzymes. The mutant genes Lt1b and Lt2 in R3202 and R3004 respectively code for feedback-desensitized isoenzymes. The presence of one of these is sufficient to allow the synthesis of methionine to overcome the growth inhibition by lysine plus threonine.     

Variability for protein and oil quality in Lupinus albus

Amino acid composition and fatty acid composition were determined on seed samples of a range of white lupin (Lupinus albus) cultivars and accessions grown in either of two environments.Variability between genotypes was found for lysine, arginine and glutamic acid content, but not for the concentrations of other amino acids. The deficiency in sulphurcontaining amino acids, typical of legume proteins, was evident, with methionine and cyst(e)ine totalling only 2.2% of the protein. Variability was limited, indicating that improvement by breeding would be impracticable. Lupinus albus differed slightly from other lupin species in amino acid composition, having higher levels of threonine, tyrosine and isoleucine, but a lower level of glutamic acid than both L. angustifolius and L. luteus. Four low-alkaloid lines of L albus each had higher lysine content than the high-alkaloid line, but ‘Kiev Mutant’, despite earlier claims, had a lysine level no higher than the other three low-alkaloid lines.Fatty acid composition of the seed oil varied considerably between genotypes. Oleic acid ranged from 43.6 to 54.4% and linolenic acid from 6.7 to 15.2%, these two fatty acids being negatively correlated at one site. Linoleic acid content varied between 17.2 and 26.9% and was not correlated with other fatty acids. Total oil content averaged 9.6% with little variability between lines.It is concluded that, relative to other lupin species, L. albus has a more favourable amino acid profile for its utilisation in cereal-based diets for animals, particularly if the energy source is wheat, which is deficient in threonine. The higher oil content would be an important energy benefit to such diets and may allow their protein/energy balance to be maintained at higher levels of incorporation of L. albus seed meal than is possible with other lupin species.     

Hepatic carbamoyl phosphate synthetase (CPS) I and urea contents in the hylid tree frog, Litoria caerulea: transition from CPS III to CPS I

The complete cDNA sequence of CPS I obtained from the liver of the hylid tree frog, Litoria caerulea, consisted of 4,485?bp which coded for 1,495 amino acids with an estimated molecular mass of 163.7?kDa. The deduced CPS I consisted of a mitochondrial targeting sequence of 33 amino acid residues, a glutaminase amidotransferase component spanning from tyrosine 95 to leucine 425, and a methylglyoxal synthetase-like component spanning from valine 441 to lysine 1566. It also comprised two cysteine residues (cysteine 1360 and cysteine 1370) that are characteristic of N-acetyl-l-glutamate dependency. Similar to the CPS I of Rana catesbeiana and Cps III of lungfishes and teleosts, it contained the Cys?CHis?CGlu catalytic triad (cysteine 304, histidine 388 and glutamate 390). All Cps III contain methionine 305 and glutamine 308, which are essential for the Cys?CHis?CGlu triad to react with glutamine, but the CPS I of R. catesbeiana contains lysine 305 and glutamate 308, and therefore cannot effectively utilize glutamine as a substrate. However, the CPS I of L. caerulea, unlike that of R. catesbeiana, contained besides glutamate 308, methionine 305 instead of lysine 305, and thus represented a transitional form between Cps III and CPS I. Indeed, CPS I of L. caerulea could utilize glutamine or NH₄ ⁺ as a substrate in vitro, but the activity obtained with glutamine?+?NH₄ ⁺ reflected that obtained with NH₄ ⁺ alone. Furthermore, only?<5?% of the glutamine synthetase activity was present in the hepatic mitochondria, indicating that CPS I of L. caerulea did not have an effective supply of glutamine in vivo. Hence, our results confirmed that the evolution of CPS I from Cps III occurred in amphibians. Since L. caerulea contained high levels of urea in its muscle and liver, which increased significantly in response to desiccation, its CPS I had the dual functions of detoxifying ammonia to urea and producing urea to reduce evaporative water loss.     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.