Nucleotides preparation

Other Nucleotides Prepare a strip of Whatman No. 2, or equivalent, filter paper about 20 x 40 cm, and draw a line across the narrow dimension about 5 cm from one end. Using... 

Lead Determine as directed under Lead Limit Test, Appendix IIIB, using a Sample Solution prepared as directed for organic compounds, and 5 xg of lead (Pb) ion in the control. Other Nucleotides Prepare a strip of Whatman No. 2, or equivalent, filter paper about 20 x 40 cm, and draw a line across the narrow dimension about 5 cm from one end. Using a micropipet, apply 10 xL of a 1 100 aqueous solution on the center of the line, and dry the paper in air. 

RNA size standards ( 20, 61, and 97 nucleotides)—prepared by the instructor Power supply Film cassette Plastic wrap Razor blade Kodak X-omat film... 

Two major metabolites were isolated by paper chromatography (from trichloroacetic acid extracts of the cells) and were identified as 8-azaguano-sine 5-phosphate and 8-azaguanosine, respectively. The two compounds were present in the ratio of 4 1. The structure of the 8-azaguanosine 5-phosphate was indicated by chromatographic and electrophoretic comparison of it with (a) the 5-nucleotide prepared by the action of snake venom on the bacterial ribonucleic acid containing 8-azaguanine, and (b) the 3-nucleotide prepared by alkaline hydrolysis. 

High specific activities of the probe can be achieved by changing to labeled nucleotide preparations of higher specific activity and reducing the labeled dNTP precursor concentration to 1-2 pM. Consequently, if the specific activity of the precursor is 1000 Ci/mmol, then 1-2 mCi should be added per ml (i.e., 25-50 pCi/25 p,l). With higher specific activities of the precursor, proportionally more radioactivity should be used to maintain the same precursor concentration (e.g., 3-6 mCi/ml if the specific activity is 3000 Ci/mmol). This can be expensive if the volume of the mixture is not scaled down, but it can be advantageous when low concentrations of probe with high specific activity are required (e.g., for certain Southern blots). The efficiency of incorporation is lower when two or more labeled nucleotides are used as precursors. 

Nucleotide solution Dilute a 100 mM GTP stock, pH 7.0, 1 1000 in L-buffer (100 /rM stock). Any vendor will supply satisfactory [a- P]GTP (specific activity >3000 Ci/mmole). Use only nucleotides prepared in water or an aqueous buffer without addition of any stabilizers such as 2-mercaptoethanol, which will inhibit the reaction. Mix 20 gd of radiolabeled nucleotide with 10 of the 100 //M GTP stock. 

Several sources of background counts are present in this assay and need to be considered. One is the free Pi in the commercially obtained isotope preparation. This background steadily increases as the nucleotide preparation ages due to spontaneous hydrolysis of the GTP and radiation damage. Another is caused by the presence of nucleotidases and nucleoside... 

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Biopolymers are the naturally occurring macromolecular materials that are the components of all living systems. There are three principal categories of biopolymers, each of which is the topic of a separate article in the Eniyclopedia proteins (qv) nucleic acids (qv) and polysaccharides (see Carbohydrates Microbial polysaccharides). Biopolymers are formed through condensation of monomeric units ie, the corresponding monomers are amino acids (qv), nucleotides, and monosaccharides, for proteins, nucleic acids, and polysaccharides, respectively. The term biopolymers is also used to describe synthetic polymers prepared from the same or similar monomer units as are the natural molecules. 

The o-nitrobenzyl and p-nitrobenzyl ethers can b prepared and cleaved by many of the methods described for benzyl ethers. The p-nitrobenzyl ether is also prepared from an alcohol and p-nitrobenzyl alcohol (trifluoroacetic anhydride, 2,6-lutidine, CH2CI2, 67% yield). In addition, the o-nitrobenzyl ether can be cleaved by irradiation (320 nm, 10 min, quant, yield of carbohydrate " 280 nm, 95% yield of nucleotide ). The p-nitrobenzyl ether has been cleaved by electrolytic reduction (—1.1 V, DMF, R4N X, 60% yield) and by reduction with Na2S204 (pH 8-9, 80-95% yield). These ethers can also be cleaved oxidatively (DDQ or electrolysis) after reduction to the aniline derivative. ... 

These were originally prepared by Khorana as selective protective groups for the 5 -OH of nucleosides and nucleotides. They were designed to be more acid-labile than the trityl group because depurination is often a problem in the acid-catalyzed removal of the trityl group. Introduction of p-methoxy groups increases the rate of hydrolysis by about one order of magnitude for each p-methoxy substituent. For 5 -protected uridine derivatives in 80% AcOH, 20°, the time for hydrolysis was... 

Nucleotide thiophosphate analogues. The preparation and purification of [ H]ATPyS, [ HJGTPyS, s ITPyS (6-thioinosine), cl ITPyS (6-chloroinosine) and [ HJATPyS are described and the general purification... 

The utility of the Zincke reaction has been extended to the preparation of various NAD and NADH analogs. Holy and co-workers synthesized a series of NAD analogs containing nucleotide bases as a means to study through-space interaction between the pyridinium and base portions. Nicotinamide-derived Zincke salt 8 was used to link with various adenine derivatives via tethers that contained hydroxyl (105 —> 106, Scheme 8.4.35), phosphonate (107—>108, Scheme 8.4.36), and carboxylate "... 

Azathymidine was first prepared by Prusoff " and the procedure was described in more detail by Hall and Haselkorn. The nucleoside was prepared here in the form of a glassy solid, but with dibenzyl-phosphochloridate it yielded a mixture of nucleotides from which crystalline 3 -phosphate, 5 -phosphate, and 3, 5 -diphosphate were prepared. 

Thus three lines of evidence define the rapidly dissociating receptor as the LR complex. Conditions known to uncouple R from G--first, guanine nucleotide and second, pertussis toxin—produce LR third, reconstitution of G protein restores receptor affinity, sensitivity to guanine nucleotide, and effector activation. In this sense, the ligand and binding behavior of this system is analogous to that of the beta-adrenergic receptor, where the LR and LRG complexes have already been studied with purified proteins and reconstituted membrane preparations (2,i0). 

Scheme 18 Preparation of nucleotide-based phosphonates via SET-induced rearrangement of allylphosphites...

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