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.     

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.     

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.     

Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.     

A new method for stoichiometric analysis of proteins in complex mixture--reevaluation of the stoichiometry of E. coli ribosomal proteins

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.     

In vivo anti inflammatory effects of the M20 IL-1 Inhibitor: I. Effects on acute inflammatory parameters

Previous studies have described an IL-1 Inhibitor produced by a myelomonocytic line developed in our laboratory (Eur J Immunol 1986; 16: 1449). This IL-1 Inhibitor was secreted by the M20 line constitutively in addition to IL-1, from which it could be separated. We have recently shown that the M20 IL-1 Inhibitor is distinct from the IL-1ra.In vitro this factor inhibited IL-1 induced proliferative responses as well as PGE₂ secretion by IL-1 induced fibroblasts. We also showed for the first time (Lymphokine Research 1988; 7(3): 268) that an IL-1 inhibitor can reduce IL-1 induced inflammatory effects. This study describes the specific effect of the M20 IL-1 Inhibitor on IL-1 induced parameters of inflammation: fever, leukocytosis and local foot pad swelling or lymph node enlargement. Purified preparations of the IL-1 Inhibitor, when injected together with IL-1, or before the IL-1, reduced fever, leukocytosis, foot pad swelling and lymph node enlargement caused by IL-1. Similar responses were obtained by injection of IL-6 or TNF, but were unaffected by the IL-1 Inhibitor, when injected together.These results indicate that the M20 IL-1 Inhibitor acts specifically on IL-1 induced responsesin vivo. The potential importance of this factor as an anti-inflammatory and immune regulatory factor, is supported by the findings of this study.Abbreviations IL-1 Interleukin 1 - IL-6 Interleukin 6 - IL-1ra Interleukin 1 receptor antagonist - TNF tumor necrosis factor     

Role of endogenous gibberellins in germination of melon (Cucumis melo) seeds

The effect of the plant growth retardants ancymidol. mefluidide and uniconazole on germination of two melon accessions differing in their ability to germinate at 14°C was examined. The accessions were the cold sensitive Noy Yizre'el and the cold tolerant Persia 202. The three growth retardants were able to delay the germination of intact Noy Yizre'el seeds, but did not affect that of intact Persia 202 seeds. On the other hand germination of decoated seeds of both accessions was unaffected by these inhibitors at normal oxygen concentration, but was inhibited at 5% oxygen. When gibberellin-like activity was measured by a dwarf rice biological assay following HPLC fractionation, it was found that seeds of Persia 202 contained much more gibberellin-like activity than Noy Yizre'el seeds. Among the extracted compounds several endogenous gibberellins were identified by combined gas chromatography-mass spectrometry (GC-MS). They included GA₄, GA₂₀, GA₁ and GA₃ in Noy Yizre'el and GA₃₄, GA₂₀, GA₁ and GA₈ in Persia 202. It is suggested that the better germination of intact Persia 202 seeds, compared to Noy Yizre'el seeds at low temperature and low oxygen concentration, is due to a higher endogenous level of GA and a better seed coat permeability to oxygen.     

Isolation and characterization of an intermediate steroid metabolite in diosgenin biosynthesis in suspension cultures of Dioscorea deltoidea cells.

The aglycon form of the steroidal sapogenin furost -5-ene-3 beta, 22,26-triol, 3 beta- chacotrioside 26 beta-D-glucopyranoside was isolated from cell suspension cultures of Dioscorea deltoidea and its molecular structure was determined by mass spectrometry and 1H and 13C n.m.r. spectroscopy. From kinetic studies and incorporation experiments with [1-14C]acetate it was concluded that the steroidal compound (in the glycoside form) is an intermediate in vivo in diosgenin biosynthesis. It accumulated in growing cells of D. deltoidea and was metabolized to diosgenin (in the glycoside form, i.e. dioscin ) in non-dividing cells.