Vectors were developed for two-step chromosomal integration of reporter genes or expression constructs. With these vectors, integration produces a disruption of the ADE8, LYS2, MET15, LEU2, HIS3 or FCY1 genes, and integrants can be easily identified by replica-plating on selective media. Integration using these 'disintegrator' vectors produces a single-copy integration of the construct of interest at the junction of the marker deletion, and removes the additional plasmid sequences. Importantly, the integrated constructs do not contain flanking sequence duplications, and therefore should be highly stable. Each of the vectors was shown to reliably integrate a TEF1-KAN expression cassette and/or GAL1-HIS3 and STE12-LacZ reporter genes.
Over time and under stressing conditions proteins are susceptible to a variety of spontaneous covalent modifications. One of the more commonly occurring types of protein damage is deamidation; the conversion of asparagines into aspartyls and isoaspartyls. The physiological significance of isoaspartyl formation is emphasized by the presence of the conserved enzyme L-isoaspartyl O-methyltransferase (PIMT), whose physiological function appears to be in preventing the accumulation of deamidated proteins. Seemingly consistent with a repair function, overexpression of PIMT in Drosophila melanogaster extends lifespan under conditions expected to contribute to protein damage. Based on structural information and sequence homology we have created mutants of residues proposed to be involved in co-factor binding in Escherichia coli PIMT. Both mutants retain S-adenosyl L-methionine binding capabilities but demonstrate dramatically reduced kinetic capabilities, perhaps suggestive of catalytic roles beyond co-factor binding. As anticipated, overexpression of the wild type enzyme in E. coli results in bacteria with increased tolerance to thermal stress. Surprisingly, even greater levels of heat tolerance were observed with overexpression of the inactive PIMT mutants. The increased survival capabilities observed with overexpression of PIMT in E. coli, and possibly in Drosophila, are not due to increased isoaspartyl repair capabilities but rather a temperature-independent induction of the heat shock system as a result of overexpression of a misfolding-prone protein.An alternate hypothesis as to the physiological substrate and function of L-isoaspartyl methyltransferase is proposed.Proteins are susceptible to a variety of spontaneous, covalent modifications that have the potential for disruption of both structure and biological activity. The formation of isoaspartyl residues, through either the deamidation of asparagines or isomerization of aspartates, are among the most rapidly occurring types of damage that afflict proteins under physiological conditions (1).The metamorphosis of asparagine and aspartate residues is initiated by the nucleophilic attack of the neighboring peptide nitrogen on the side chain carbonyl, resulting in the cyclization of the side chain with the main chain to the formation of a succinimide ring (Fig.
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.
We have developed plasmid vectors to enable selection of diploids from mating reactions between haploid strains that lack compatible recessive genetic markers. The plasmids bear one of five different dominant selectable markers, kanMX4, hphMX4, natMX4, patMX3 or ZEO, a yeast origin of replication, and the URA3 gene. Diploids can be selected from mating reactions between haploids transformed with plasmids expressing different dominant markers, by using a combination of drugs that select for both markers. Following non-selective growth, diploids that subsequently become cured of the plasmids can be directly selected on 5-FOA, for ura3 auxotrophs, or identified by replica-plating onto appropriate selective media.
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