Labeling uniform incorporation

Nick translation widely used classical method producing probes of varying lengths and labels uniformly incorporated requires ds DNA amount of DNA decreases during labeling... 

Microbiological procedures which exploit the ability of bacteria and photosynthetic algae to incorporate exogenous labeled precursors such as 14CO2, SO%, and 32pQ3- [ can be used to label complex molecules in cells such as proteins (qv) and nucleic acids (qv), which are then processed to give labeled constituents such as uniformly labeled C-amino acids, C-nucleotides, C-fipids, LS-amino acids, etc (8). 

For example, cells of E. coli can be grown on a minimal medium containing [15N] NH4C1. Since 13C can also be introduced in a similar way it is possible to incorporate both isomers simultaneously. Production of uniformly labeled protein containing 15N and / or 13C provides the basis for multidimensional isotope-edited spectra necessary for protein structure determination (next section) and for study of tautomerization of histidine rings (Eq. 2-6) 460/462-464 15N chemical shifts of groups in proteins are spread over a broad range (Table 3-3).465... 

This enzymically prepared product was, therefore, tested with bacterial extracts. Almost quantitative conversion to shikimate took place. Furthermore, D-olheptulose diphosphate labeled in carbon atoms 4,5,6, and 7 with C, prepared from uniformly labeled D-oZiro-heptulose 7-phosphate and unlabeled n-fructose diphosphate (see Fig. 3), was converted to shikimate labeled exclusively in carbon atoms 3,4,5, and 6. It is clear, however, that a cyclization of the intact carbon-chain of D-aZtriose phosphate isomerase, carbon atoms 1,2, and 3 of the heptulose diphosphate would be derived from G-(l,6), G-(2,5), and G-(3,4), respectively. Carbon atoms 7,1, and 2 of shikimate, as discussed previously (see Fig. 1), are derived from the reverse sequence, namely, G-(3,4), G-(2,5), and G-(l,6). Apparently, carbon atoms 1,2, and 3 of the heptulose diphosphate become detached, and their order is reversed, before their incorporation into shikimic acid. 

The MVA pathway was accepted as the unique biosynthetic pathway for the formation of aU isoprenoids in aU living organisms. Discrepancies with this general assertion appeared, however, as early as the 1950s (1, 2). For instance, -labeled MVA was not incorporated into chloroplast isoprenoids (e.g., carotenoids 25 and phytol 24 from chlorophylls Fig. 6), whereas it was well incorporated into phytosterols 27 synthesized in the cytoplasm. Unexpected labeling patterns were found in the prenyl chain of ubiquinone 22 in Escherichia coli at incorporation of C-labeled acetate. Finally, the labeling pattern in an isoprene unit from the sesquiterpenic pentalenene 21 series from a Streptomyces species at incorporation of uniformly... 

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

Labeled carbon. Succinate uniformly labeled with is added to cells actively engaged in pyrimidine biosynthesis. Propose a mechanism by which carbon atoms from succinate could be incorporated into a pyrimidine. At what positions is the pyrimidine labeled ... 

Recombinant human leptin was recently cloned (8) and expressed in E. coli, and demonstrated to effectively regulate adiposity in mice through modulation of appetite and metabolism (9, 10). The molecule contains four methionine residues at positions 1, 54, 68, and 136. In this paper, we report the separation and characterization of three norleucine-incorporated recombinant human leptins which were uniformly labeled with 15n isotope or double labeled with and isotopes. The extent of incorporation at each methionine residue can be determined by reverse-phase HPLC and amino acid analysis methods. The norleucine incorporation was observed preferentially occurring at the internal Met residues. 

Spenser and co-workers 123) have investigated the biosynthesis of berberine and related alkaloids elaborated by Hydrastis canadensis L. In separate feeding experiments, D-glucose-i C (uniformly labeled), DL-phenylalanine-2-i4C, DL-tyrosine-2-i4C DL-tyrosine-S-i C, and 3,4-dihydroxy-2-phenylethylamine-l-i4C (dopamine) were administered to the growing plants. Of the compounds tested tyrosine was the most efficient precursor of the major alkaloids, berberine and hydrastine, and dopamine was almost as good. Glucose was a much less efficient precursor, and the incorporation of phenylalanine into these alkaloids was almost negligible. 

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