The budding yeast Saccharomyces cerevisiae differentiates into filamentous invasively growing forms under conditions of nutrient limitation. This response is dependent on the transcription factor Ste12 and on the mating pheromone-response mitogen-activated protein (MAP) kinase cascade, but a mechanism for regulation of Ste12 by nutrient limitation has not been defined. Here we show that Ste12 function in filamentous growth is regulated by the cyclin-dependent kinase Srb10 (also known as Cdk8), which is associated with the RNA polymerase II holoenzyme. Srb10 inhibits filamentous growth in cells growing in rich medium by phosphorylating Ste12 and decreasing its stability. Under conditions of limiting nitrogen, loss of Srb10 protein and kinase activity occurs, with a corresponding loss of Ste12 phosphorylation. Mutation of the Srb10-dependent phosphorylation sites increases pseudohyphal development but has no effect on the pheromone response of haploid yeast. Srb10 kinase activity is also regulated independently of the mating pheromone-response pathway. This indicates that Srb10 controls Ste12 activity for filamentous growth in response to nitrogen limitation and is consistent with the hypothesis that Srb10 regulates gene-specific activators in response to physiological signals to coordinate gene expression with growth potential.
A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.
Aerolysin, the hemolytic toxin produced by Aeromonas hydrophila, has been purified by a combination of salt fractionation, gel filtration, and ion-exchange and hydroxyapatite chromatography. The resulting protein has a molecular weight of 51 500 and appears homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. It is free of detectable protease and phospholipase activities. The purified protein can be separated into two active components with pIs of 5.39 and 5.46 by isoelectric focusing. Both components are found in the original culture supernatant indicating that the multiplicity is not due to proteolysis during isolation. Purified aerolysin is unstable even at 25 degrees C and its hemolytic action is inhibited by certain reducing agents including ferrous iron and cysteine. It appears to be the only toxin hemolytic to human cells that is produced by A. hydrophila under the conditions described.
The repressed transactivator (RTA) yeast two-hybrid system was developed to enable genetic identification of interactions with transcriptional activator proteins. We have devised modifications of this system that enable its use in screening for inhibitors of protein interactions from small molecule compound libraries. We show that inhibition of protein interactions can be measured by monitoring growth in selective medium containing 3-aminotriazole (3-AT) and using this assay have identified inhibitors of four independent protein interactions in screens with a 23,000 small molecule compound library. Compounds found to inhibit one of the tested interactions between FKBP12 and the transforming growth factor beta receptor (TGFbeta-R) were validated in vivo and found to inhibit calcineurin-dependent signaling in T cells. One of these compounds was also found to cause elevated basal expression of a TGFbeta-R/SMAD-dependent reporter gene. These results demonstrate the capability of the RTA small molecule screening assay for discovery of potentially novel therapeutic compounds.
A comparison of the phenotypic properties of three rubella vaccines (HPV77/DE5, RA27/3 and Cendehill) and four wild-type (wt+) isolates (M33, Therien, Thomas and IB2) has been carried out. Differences in growth characteristics, plaque morphology and temperature sensitivity were identified. In addition differential reactivity of the strains to polyclonal and a monoclonal anti-E1 antibody were found in immunoperoxidase-staining reactions. The ability of the wt+ and vaccine strains to infect lymphoreticular cells and chondrocytes, also varied in that the RA27/3 and Cendehill strains were highly restricted in both these cell types while the wt+ strains and HPV77/DE5 vaccine grew to high titer. This biological variation was associated with differences in E1 and E2 glycoproteins detected on immunoblots.
The effect of the induction of the enzymes of the phosphorylated pathway of L-serine biosynthesis on the thermodynamic relationships among the reactions has been determined in rat liver in vivo. The mass action ratios of the reactions involved were calculated from the concentrations of appropriate metabolites in freeze-clamped liver from animals fed normal and low-protein diets for 2 weeks. These ratios were compared with the equilibrium constants of the same reactions previously determined under physiological conditions and the results previously obtained in the rabbit. The thermodynamic relationships in the pathway were different between the normal rat and rabbit as might have been expected, because of the significantly lower activities of the L-serine biosynthetic enzymes in the former animal. Although the delta G for the overall pathway is nearly identical in the rat and rabbit (-5.8 versus -5.5 kcal/mol, respectively), the distribution of delta G among the reactions is different. The disequilibrium in the pathway in rat liver is nearly equally divided between the L-phosphoserine phosphatase (EC 3.1.3.3) step and the other two reactions [D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) and L-phosphoserine aminotransferase (EC 2.6.1.52)], whereas in rabbit the phosphatase reaction accounts for nearly the entire delta G. Feeding the rat a low protein diet, however, induced the activity of D-3-phosphoglycerate dehydrogenase 12-fold, that of L-phosphoserine aminotransferase 20-fold, and that of L-phosphoserine phosphatase 2-fold. With the induction of the pathway, L-phosphoserine appeared in the tissue, there was a more than 3-fold rise in L-serine in the liver, and the pattern of delta G in the rat liver approached that in the rabbit.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.