Network-Free Inference of Knockout Effects in Yeast

Perturbation experiments, in which a certain gene is knocked out and the expression levels of other genes are observed, constitute a fundamental step in uncovering the intricate wiring diagrams in the living cell and elucidating the causal roles of genes in signaling and regulation. Here we present a novel framework for analyzing large cohorts of gene knockout experiments and their genome-wide effects on expression levels. We devise clustering-like algorithms that identify groups of genes that behave similarly with respect to the knockout data, and utilize them to predict knockout effects and to annotate physical interactions between proteins as inhibiting or activating. Differing from previous approaches, our prediction approach does not depend on physical network information; the latter is used only for the annotation task. Consequently, it is both more efficient and of wider applicability than previous methods. We evaluate our approach using a large scale collection of gene knockout experiments in yeast, comparing it to the state-of-the-art SPINE algorithm. In cross validation tests, our algorithm exhibits superior prediction accuracy, while at the same time increasing the coverage by over 25-fold. Significant coverage gains are obtained also in the annotation of the physical network.     

Phaseollin production in cell suspensions and whole plants of Phaseolus vulgaris

Production of phaseollin was measured in cell suspension cultures and whole plants of Phaseolus vulgaris. In suspension cultures phaseollin appeared when there was no further increase in cell mass. Cells transferred to a medium without auxins yielded three times higher phaseollin concentrations than cells grown in their presence. Addition of autoclaved fungal mycelia or polysaccharides as elicitors resulted in an increased phaseollin concentration in the cell suspension.In whole plants phaseollin could be detected only after the plants were challenged by a fungus which caused lesions (browning) of the upper root neck region, Rhizoctonia solani. Treatment of non-infected plants with autoclaved fungal mycelia or other elicitors did not induce phaseollin production. However, when they were added before or together with the pathogenic fungus, the elicitors further increased phaseollin concentration in the root neck regions of the plants. This indicated that the pathogenic fungus was important for the penetration of the elicitors to inner plant tissues where phaseollin (and probably other phytoalexins) is produced.     

The effect of acute and chronic ethanol administration on serum corticosterone concentration in rats

The effect of intraperitoneally (i.p.) and intragastrically (i.g.) administered ethanol solution, and the influence of voluntary ethanol uptake (20% v/v) on adrenocortical activity of adult male rats was studied. Both i.p. and i.g. ethanol administration resulted in a significant activation of adrenocortical mechanisms, while voluntary ethanol uptake failed to induce elevation of serum corticosterone concentration. No difference was found in blood ethanol concentration among these groups. The responsiveness of adrenocortical mechanisms was also tested in rats which were given the free choice between ethanol solution (5% v/v) and tap-water for three weeks. Unavoidable electric foot-shocks, as stressor, resulted in an elevation of serum corticosterone concentration in control animals, but this response was found to be significantly reduced in chronically ethanol drinking rats.     

The primary structure of rat ribosomal protein L7. The presence near the amino terminus of L7 of five tandem repeats of a sequence of 12 amino acids

The covalent structure of rat ribosomal protein L7 was determined in part from the sequence of nucleotides in a recombinant cDNA and in part from the sequence of amino acids in portions of the protein. The complementary analyses supplemented and confirmed each other. Ribosomal protein L7 contains 258 amino acids and has a molecular weight of 30,040. The protein has an unusual and striking structural feature near the NH2 terminus: five tandem repeats of a sequence of 12 residues. Rat L7 appears to be related to ribosomal protein L7 from the moderate halophile Vibrio costicola and perhaps to L30 from Bacillus stearothermophilus, to L7 from the moderate halophile NRCC 41227, and to L22 from Nicotinia tobaccum chloroplast. In addition, there is a sequence of 24 amino acids in rat protein L7 that may be related to segments of the same number of residues in Escherichia coli ribosomal proteins S10, S15, L9, and L22.     

Isoenzyme pattern and subcellular localisation of enzymes involved in fumaric acid accumulation by Rhizopus oryzae

Summary Electrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

Morphine induced thyroxine release from rat thyroid gland in vitro

Male rat thyroid glands were incubated for two hours in Krebs-Ringer bicarbonate buffer with different amounts of morphine and/or naloxone. Five micrograms/ml morphine produced a significant increase in the T4 concentration of incubation medium, and resulted in an accumulation of cAMP in the tissue. Naloxone did not change the T4 release but its incubation with morphine prevented the morphine-induced changes. Similarly, naloxone inhibited the morphine-induced accumulation of cAMP in the thyroid tissue.     

3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate. A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase

A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP. Benzoylbenzoyl-[5-3H]CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc. E. coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-[alpha-32P]CTP as a photoaffinity label. This specific covalent binding occurred using enzyme preparations of different degrees of purity. These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes. These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides.