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 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.     

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.     

Inducible overexpression of the FUM1 gene in Saccharomyces cerevisiae: localization of fumarase and efficient fumaric acid bioconversion to L-malic acid

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

Physical mapping of plastid DNA variation among eleven Nicotiana species

Summary Plastid DNA of seven American and four Australian species of the genus Nicotiana was examined by restriction endonuclease analysis using the enzymes Sal I, Bgl I, Pst I, Kpn I, Xho I, Pvu II and Eco RI. These endonucleases collectively distinguish more than 120 sites on N. tabacum plastid DNA. The DNAs of all ten species exhibited restriction patterns distinguishable from those of N. tabacum for at least one of the enzymes used. All distinctive sites were physically mapped taking advantage of the restriction cleavage site map available for plastid DNA from Nicotiana tabacum (Seyer et al. 1981). This map was extended for the restriction endonucleases Pst I and Kpn I. In spite of variation in detail, the overall fragment order was found to be the same for plastid DNA from the eleven Nicotiana species. Most of the DNA changes resulted from small insertions/deletions and, possibly, inversions. They are located within seven regions scattered along the plastid chromosome. The divergence pattern of the Nicotiana plastid chromosomes was strikingly similar to that found in the genus Oenothera subsection Euoenothera (Gordon et al. 1982). The possible role of replication as a factor in the evolution of divergence patterns is discussed. The restriction patterns of plastid DNA from species within a continent resembled each other with one exception in each instance. The American species N. repanda showed patterns similar to those of most Australian species, and those of the Australian species N. debneyi resembled those of most American species.Abbreviations ims isonuclear male sterile - ptDNA plastid chloroplast DNA - Rubisco ribulosebisphosphate carboxylase/oxygenase - kbp kilobase pairs - LSU large subunit of Rubisco     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.