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.     

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.     

Functional Changes Associated with Structural Alterations Induced by Mobilization of a P Element Inserted in the Sex-lethal Gene of Drosophila

Genetic analysis of rearrangements within the multifunctional sex determining gene Sex-lethal has allowed correlation of changes in specific functions with DNA alterations. Rearrangements were isolated by mobilization of a P element which is on the 5' side of the gene, at coordinate 0. Previous work has shown that rearrangements associated with alterations in Sxl gene function are found within an 11-kb region between coordinates-11 and 0. Here it is shown that insertion of foreign DNA, per se, at coordinate 0 is compatible with wild-type gene function. However, deletion of sequences on either side of this point generates a mutant phenotype. Deletions extending distally beyond coordinate -6.5 kb result in a null phenotype, whereas smaller distal deletions or proximal deletions eliminate only some Sxl functions.     

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.     

Phytotoxins from Alternaria helianthi: radicinin,and the structures of deoxyradicinol and radianthin

A novel compound, radianthin, with phytotoxic activity was isolated from liquid cultures of Alternaria helianthi and identified as a pyrone related to radicinin. A second metabolite was identified as radicinin itself while deoxyradicinol is described for the first time as a natural product.