Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.
Thes ynthesis of oligosaccharidesu sing mutant glycosidases hasb een dynamically developing due to the need for novelc arbohydrate-based materials.C hitooligomers (b-1!4-linkedo ligomers of N-acetylglucosamine) are bioactive compounds applicablei nm any industrial andp harmacological areas;h owever, their accessibility is still ratherl ow. In this work, GH20 b-N-acetylhexosaminidase from the fungus Talaromyces flavus wase ngineered by site-directed mutagenesis to obtain three efficiently transglycosylating variants with ca. 200-timess uppressedh ydrolytic activity.T hus,w eh ave prepared the first GH20 transglycosidases.Inthe reactions cat-alyzed by these mutant b-N-acetylhexosaminidases we were able to easily prepare andi solate bothn atural and modified chitooligomers in sufficient amountsf or their complete spectral characterization and possible further application. Thep resented method for the synthesis of chitooligomers with aglycones suitablef or linkingt oo ther biological structures is simple and robust enough to be easily scaled up.
A two-phase cultivation system was developed which will enable studies of streptomycete differentiation by molecular biological and global techniques such as transcriptomics and proteomics. The system is based on a solid phase formed by glass beads corresponding to particles in soil, clay, or sand natural habitats of streptomycetes. The beads are immersed in a liquid medium that allows easy modification or replacement of nutrients and growth factors as well as radioactive labeling of proteins. Scanning electron microscopy was used to analyze morphological differentiation of streptomycetes on glass beads and two-dimensional protein electrophoresis to demonstrate the potential of the system for analyses of protein synthesis profiles during the developmental program. This system facilitates studies of differentiation including expression and posttranslation modifications of streptomycetes proteins, secondary metabolite biosynthesis, and morphological development.Streptomycetes are saprophytic filamentous gram-positive bacteria inhabiting particulate soil ecosystems and marine sediments throughout the world. They are a particularly interesting group of bacteria, having a complex life cycle, diverse metabolic capabilities, and one of the largest of bacterial genomes (2). They excrete extracellular enzymes to hydrolyze polymers such as starch and cellulose and utilize the soluble breakdown products. Their ability to produce many important antibiotics and other useful metabolites has been of interest to academics and exploited by many pharmaceutical companies. The life cycle of streptomycetes is initiated by spore germination followed by the development of a branched hyphal network covering the surface of soil particles or organic debris. In response to complex but still poorly defined signals, the substrate mycelium produces aerial hyphae that eventually undergo septation to yield chains of unigenomic spores. This has proven to be a complex process linked to primary metabolism (17) and production of secondary metabolites, including many antibiotics (5, 6, 9, 18). Therefore, it is important to study streptomycete morphological differentiation, ideally under conditions that mimic its natural environment as closely as possible. To achieve this, streptomycetes have been cultivated on agar plates, sometimes covered with sheets of cellophane (1, 7) to facilitate harvesting of cells. However, there are several disadvantages. Agar gels do not simulate the particulate nature of soil ecosystems. Furthermore, it is not well adapted to allowing adjustment of the medium composition during growth, to providing radiolabeled nutrients or growth factors in a controllable manner, or to analyzing secondary metabolite production during cell differentiation. The problem of obtaining large homogenous samples from differentiating streptomycetes in a reproducible manner is encountered in all the transcriptomic and proteomic studies.Here, we present a novel system for cultivation of the mycelial bacteria in a two-phase system composed of glas...
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