Photoaddition of trimethylpsoralen as a probe for the intracellular organization of Escherchia coli DNA.

It has been previously demonstrated that photoreaction with 4,5',8-tri-methylpsoralen (trioxsalen) can be used as a probe for the in vivo structure of eucaryotic chromatin. We have used this probe to analyze the organization of intracellular Escherichia coli DNA. In contrast to eucaryotic DNA, bacterial DNA within the intact cell is not protected from saturating doses of trioxsalen photoaddition. The final level of covalently bound trioxsalen upon saturation is identical to that found with purified DNA. In addition, the distribution of interstrand DNA cross-links formed by low doses of trioxsalen photoadducts does not exhibit the repeating pattern that has been observed with eucaryotic nucleosomes.     

Carbamyl phosphate synthetase A of Neurospora crassa.

Carbamyl phosphate synthetase A of Neurospora crassa was partially purified from mitochondrial extracts. It is an extremely unstable enzyme (t 1/2 = 45 min at 25 detrees C) made up of two unequal subunits. The native enzyme has a molecular weight of approximately 175,000, and the large subunit has a molecular weight of about 125,000. Both the native enzyme and its large subunit are quite asymmetric, as revealed by slow sedimentation in sucrose gradents (7.3S and 6.6S, respectively). The small subunit has not been identified physically as a separate entity. The denaturation of the native, glutamine-dependent activity is correlated with dissociation of subunits, the larger of which retains a more stable, ammonia-dependent activity. Neither substrates nor any other agents except glycerol or polyethylene glycol appreciably stabilized the glutamine-dependent activity. Kinetic studies showed the native enzyme to have a Km for glutamine of about 0.16 mM, and a Km for NH4Cl of about 16 mM, at the optimal pH, 8.0. The enzyme, using either N donor, has a K+ requirement for activity, for which NH4+ can substitute. The glutamine leads to glutamate reaction, which requires the small subunit, also requires the large subunit and all reaction substrates for optimal activity. Other evidences of subunit interaction are the greater activity of the native enzyme, as opposed to the large subunit, with low concentrations of adenosine 5'-triphosphate-Mg2+, and in the stimulation of the ammonia-dependent activity of the native enzyme by glycine. Curiously, although the enzyme's role in biosynthesis is confined to the arginine pathway, it is completely indifferent to arginine or its precursors as feedback effectors or activators. The enzyme is compared with carbamyl phosphate synthetases of other organisms.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.     

Development of alfalfa strains with differential tolerance to aluminum toxicity

Summary Aluminum toxicity limits root growth in acid subsoils that are difficult to lime. An alternative to subsoil liming is the development of plants having greater tolerance to Al. Alfalfa (Medicago sativa L.) is considered an Al-susceptible species. Preliminary studies indicated that alfalfa cultivars differ in Al tolerance, but the extreme plant-to-plant variation within cultivars prevented the establishment of clearcut cultivar differences.Tolerant and susceptible plants were selected from each of six cultivars (DuPuits, Atlantic, Team, Buffalo, Grimm, and Sirsa 9) grown on an Al-toxic Bladen soil at pH 4.1 to 4.3. The tolerant selections were repotted and interpollinated to form one population of polycross seed. Susceptible selections were treated similarly to form a second population. These two populations, tolerant and susceptible, were subjected to an additional cycle of recurrent phenotypic selection for tolerance and susceptibility, respectively, to Al-toxic Bladen soil at pH 4.6.Plants from the population selected for tolerance to the acid Bladen soil were significantly higher in both root and top vigor on Al-toxic Tatum soil than plants from the population selected for susceptibility. The results indicated that Al tolerance is a heritable trait in these alfalfa populations and that recurrent selection can be used effectively to develop strains having differential tolerance to Al-toxic soils. The observation that only 2% of the plants from the tolerant population were in the most tolerant class suggests a good opportunity for more progress in selecting toward Al tolerance.     

Biosynthesis of wound ethylene in morning-glory flower tissue

Production of wound ethylene was investigated in rib segments excised from flower buds of morning-glory (Ipomoea tricolor). Segments of the ribs were cut from buds 2 days before flower opening, floated overnight on 5 mm KCl solution, and transferred to agar the following morning. These immature segments evolved only a small quantity of ethylene during incubation on agar, with most of the production occurring in the morning. When such segments were wounded mechanically early in the afternoon, the rate of ethylene production rose more than 10-fold within 1 hour and returned to a low rate after about 3 hours.Production of ethylene by both untreated and wounded rib segments was inhibited more than 95% by overnight pretreatment with the ethoxy analog of rhizobitoxine (3 x 10(-5) and 10(-4)m). After overnight exposure of segments to 9 muml-methionine-U-(14)C, the specific radioactivity of the ethylene evolved by untreated and wounded tissue was determined and compared to the specific radioactivities of carbon atoms 3 plus 4 of methionine and S-methylmethionine (SMM) extracted from the segments. The specific radioactivity of methionine was about one-half that of SMM; neither value was significantly affected by wounding. The specific radioactivity of ethylene evolved by untreated tissue was close to that of SMM. In wounded tissue the specific radioactivity of the ethylene evolved was lower, but still above that of methionine. These results are consistent with the interpretations that wound ethylene is synthesized from carbon atoms 3 plus 4 of either SMM or methionine. On the basis of earlier experiments with senescing rib segments, it is suggested that methionine serves as the precursor of the wound ethylene.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) in adipose tissue in vivo by glucocorticoids and insulin.

1. The regulation of the synthesis of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in epididymal adipose tissue, liver and kidney in vivo was studied immunochemically. 2. Phosphoenolpyruvate carboxykinase (GTP) synthesis in adipose tissue is increased by starvation, diabetes and noradrenaline, and decreased by re-feeding and insulin. These changes were also seen in adrenalectomized rats and are qualitatively similar to those observed for the liver enzyme. This indicates the involvement of cyclic AMP as an inducer and insulin as a de-inducer in the regulation of phosphoenolpyruvate carboxykinase (GTP) in both tissues. (Induction and de-induction are defined as selective increase and decrease respectively in the rate of enzyme synthesis, regardless of the mechanism involved.)3. Adrenalectomy had little effect on phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney, but increased the synthesis rate of the adipose-tissue enzyme. Starvation and adrenalectomy had additive effects in increasing the synthesis rate of adipose-tissue phosphoenolpyruvate carboxykinase (GTP). In adrenalectomized diabetic rats glucocorticoids increased phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney while decreasing enzyme synthesis in adipose tissue. De-induction of adipose tissue phosphoenolpyruvate carboxykinase (GTP) is therefore regulated independently by glucocorticoids and insulin. 4. Although liver, kidney and adipose-tissue phosphoenolpyruvate carboxykinases (GTP) are seemingly identical, there is an apparent tissue-specific differentiation in regulatory systems for the enzyme.     

Ion dependence of the tarsal sugar receptor of the blowfly Phormia regina.

The activity of the tarsal sugar receptor is greatly reduced following prolonged water exposure. The animal's behavior, which characteristícally reflects receptor input, also shows decreased acceptance of sucrose solutions following prolonged tarsal immersion in deionized water. Long exposure of the tarsi to Bodenstein's saline instead of water does not produce as large a decrement in the acceptance response as does water exposure.Recovery of the behavioral response occurs spontaneously after a few hours. The original response level can also be restored immediately if a moderate concentration (0.05 to 0.2 M) of KCl or NaCl is added to the sucrose stimulus. The effect of LiCl is ambiguous: it inhibits the normal sucrose response, thereby tending to mask any restorative effects. The electrophysiological data show that the cellular response level is also restored when Na⁺ or K⁺ ions are present in the stimulus.The above data are interpreted to mean that the effect of tarsal water exposure is to slowly leach out ions in the effective extracellular fluid surrounding the receptor membrane, thus lowering the membrane potential and deceasing the receptor potential upon stimulation. The fact that Na⁺ and K⁺ when supplied in the stimulating solution temporarily restore the original response level suggests that these extrinsically added ions can be used as current carrying ions to depolarize the cell. The data suggest that the sensillum contains three functional compartments interconnected by partial diffusion barriers: (1) a ‘receptor compartment’ (2) an axial cylinder which contains the dendrites and functions as the immediate extracellular ion source, and (3) a larger axial cylinder which serves as an ion reservoir.A method for statistically analyzing behavioral acceptance data is presented.     

Extended Monod equation for batch cultures with multiple exponential phases

The batch growth curves of Laclobacillus delbreuckii exhibit several exponential phases. From the results of a series of shaker flask experiments, the position of the slope changes in the growth curve and the overall bacterial yield is affected by the initial amount of yeast extract in the medium. It is postulated that this behavior is due to several growth enhancing substances that are initially in the yeast extract and are consumed by the bacteria during the course of the fermentation. Using a Monod-type expression to represent the effect of growth enhancing components in a proposed growth rate expression, a mathematical model of the system is set up and solved on the analog computer.     

Hormonal regulation of hepatic P-enolpyruvate carboxykinase (GTP) during development.

Hepatic gluconeogenesis in the rat does not begin until birth. The enzyme P-enolpyruvate carboxykinase appears initially at birth and is the final enzyme in the gluconeogenic sequence to develop. The appearance of this enzyme in the cytosol of rat liver is caused by the stimulation of enzyme synthesis, probably due directly to an increase in the hepatic concentration of cAMP. Enzyme degradation does not begin until 36 hours after birth. Studies with fetal rats in utero have shown that dibutyryl cAMP or glucagon will stimulate P-enolpyruvate carboxykinase synthesis and that this effect can be blocked by insulin. Insulin is known to depress the synthesis of P-enolpyruvate carboxykinase in adult rat liver and in Reuber H-35 liver cells in culture. The glucocorticoids are without effect on the synthesis of the enzyme in fetal rat liver. Work by Girard et al. (J. Clin. Invest. 52: 3190, 1973) has established that the molar ratio of insulin to glucagon drops from 10 immediately after birth, to 1 after one hour. This is due to both a rise in glucagon and a fall in insulin concentrations at birth. These studies, together with our work on the synthesis of P-enolpyruvate carboxykinase, indicate that the sharp drop in the concentration of insulin may relieve the normal inhibition of enzyme synthesis. This would allow the initial stimulation of enzyme synthesis by the glucagon-mediated rise in the concentration of CAMP.     

Serine transhydroxymethylase isoenzymes from a facultative methylotroph.

Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.