Previous methods for the preparation of phosphorothioate-containing oligodeoxyribonucleotides rely on the reaction of phosphite triesters with sulfurizing reagents such as tetraethylthiuram disulfide (TETD) and 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent). However, these and other sulfurizing reagents suffer from several disadvantages, and there is great impetus for the development of improved methods for sulfur transfer that are fully compatible with standard automated DNA synthesis. The present report describes the use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents that meet these needs. Both reagents are easily prepared, and are stable upon prolonged room temperature storage in acetonitrile solution. The reagents are used at low concentrations (0.05 M) and for short reaction times (30 s). The methodology has been proven for the automated synthesis on 0.2-1.0 micromol scales of oligodeoxyribonucleotides, of length 6-20 bases, containing the phosphorothioate substitution at either a single site or at all positions.
The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins. Src homology (SH)1 domains are building blocks in many proteins involved in intracellular signal transduction. Detailed understanding of the pathways involving these domains is complicated by the substantial range of individual specificities in SH2 and SH3 domains, and their combination into large proteins that can form multiple homo-and heteromolecular associations. The reductionist approach of studying individual domains has been very successful (1-3). Nonetheless, the interactions between the domains are still poorly understood. These interactions are likely to be of significance in explaining more fully the complete activities of the signal-transducing complexes. A detailed structural picture of the inter-and intramolecular organization of the domains is then a significant objective. Several cases of multiple SH2 and SH3 domaincontaining constructs have been studied structurally (Lck SH(32) (Ref. 4), Grb2 SH(323) (Ref. 5), and Abl SH(32) (Ref. 6)), but there are technical limitations to current structural approaches. In the crystalline state, packing forces may be of the same magnitude as the weak interdomain forces, so there are limits to the interpretation of diffraction studies. Solution studies by NMR are only applicable in a molecular mass range less than ϳ30 kDa. For NMR, time-averaged NOE constraints in rapidly exchanging conformations are not readily interpretable. As a complement to direct structural methods, we propose the investigation of such multidomain complexes using "consolidated" ligands. These ligands, having multiple binding portions, may be expected to bind with high affinity and specificity when a linker between the two affine segments is of the correct length, and there is little affinity of the linker itself. The consolidated ligands do not necessarily resemble any natural ligand. Such ligands are demonstrated here, binding to the SH(32) of Abelson kinase. It is reasonable to assume that consolidated ligands for dual SH2, dual SH3, or multiple combinations with other ligands can be produced using a similar approach.The proposed consolidated ligands are similar in concept to affinity reagents, with the modification that the second functionalit...
The Per-ARNT-Sim (PAS) domain serine/threonine kinase PAS kinase is involved in energy flux and protein synthesis. In yeast, PSK1 and PSK2 are two partially redundant PASK homologs. We recently generated PSK2 deletion mutant and showed that Psk2 acts as a nutrient-sensing protein kinase to modulate Ultradian clock-coupled respiratory oscillation in yeast. Here, we show that deletion of PSK1 increased the sensitivity of yeast cells to oxidative stress (H2 O2 treatment) and partially inhibited cell growth; however, the growth of the PSK2-deleted mutant was similar to that of the wild type. Superoxide dismutase-1 (SOD1) mRNA and protein levels were lower in PSK1-deletion mutant than the wild type. The mRNA levels of stress response genes CTT1, HSP104, ATH1, NTH1 and SOD2 were similar in both the PSK1-deleted mutant and wild-type yeast. Furthermore, intracellular accumulation of reactive oxygen species (ROS) was noted in PSK1-deleted mutant. These results suggest that PSK1 induces SOD1 expression to protect against oxidative stress in yeast.
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.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.