Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.     

Microbial production of d-amino acids

The production of d-aminoacylase by Alcaligenes denitrificans and Alcaligenes faecalis has been studied. The enzyme was inducibly produced and N-acetyl-d-leucine and N-acetyl-d-valine were the most effective inducers. d-methionine, d-valine, d-phenylalamine and d-leucine were produced by the enzymic hydrolysis of the appropriate N-acetyl-d-amino-acids with whole cell biomass. The hydrolysis of N-acetyl-d-methionine by A. denitrificans and N-acetyl-d-valine by A. faecalis was preferential. Maximum yields of d-methionine and d-valine were 94.3 and 84.7% at a specific product formation rate of 20.10 and 19.19 μmol min⁻¹ mg⁻¹ of wet cells at 20 mM substrate concentration and 5 mg ml⁻¹ of cell density.     

Correlation studies on yield and fibre traits in upland cotton (Gossypium hirsutum L.)

Summary Two diverse parents of upland cotton namely J.34 and I.C. 1926 were crossed. A comparison between biparental intermated progenies and F₃ families indicated alteration of correlation coefficient between yield and halo length. The significant negative correlation in F₃ population between these two attributes changed to a positive but non significant one in biparental intermated progenies. A change in correlation coefficients was expected due to breakage of linkage upon intermating. An increase in the correlation coefficients could also be expected when linkages are predominantly in the repulsion phase. It is suggested that intermating in early generations coupled with selection of desirable segregants may prove a useful method for improving yield and quality simultaneously. The diallel selective mating system may also supplement intermating to improve yield and quality in cotton.Part of Ph.D. Thesis submitted to the Haryana Agricultural University. Hissar-125004, India     

Nucleolar organizer region heteromorphism associated with trisomy-21: a risk factor for non-disjunction?

An unusual nucleolar organizer region (double NOR) on chromosome 13 was observed in a Down syndrome child [47, XY, +21, dNOR(13)]. The variant chromosome was inherited from the mother [46, XX, dNOR(13)]. The extra chromosome 21 in the proband was maternal origin. The frequency of NOR chromosome association showed relatively high frequency in the mother and proband as compared to the controls. The result suggest that chromosome variants involving extra copies of NOR may indeed be involved in the meiotic nondisjunction of chromosome-21.     

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.     

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.     

Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic L-alanine: 4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial L-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of L-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial L-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of L-alanine: 4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment.     

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

Batch and multistage continuous ethanol fermentation of cellulose hydrolysate and optimum design of fermentor by graphical analysis

Batch and single-flow four-stage continuous ethanol fermentations of bagasse hydrolysate have been investigated at pH 4.0 and 30°C with a strain of the yeast Saccharomyces cerevisiae. The studies were carried out in the laboratory four-stage cascade continuous stirred-tank fermentors at varying feed glucose concentrations (10, 14, 18, and 22%). The range of dilution rates employed varied from 0.05 to 0.2 hr^?1. The hydrolysate was supplemented with a cheap nitrogen source (CNS), CaCl₂·H₂O and MgSO₄·7H₂O. A 2% (v/v) CNS concentration was found to be sufficient to avoid growth limitation at a glucose concentration of 116 g/liter. The conditions of continuous culture in a multistage system are predicted by a graphical method based on batch-culture data. The results thus obtained are compared with those predicted by kinetic models and with the experimental results. The variations between the results obtained experimentally and those computed either by a kinetic model or by graphical analyses were found to be within the limits of experimental error. The solutions based on the concept of minimum residence time necessary to achieve the desired biomass or product concentrations are also discussed.