Nucleotides synthetic

A prototype bDNA assay was developed for quantification of HGV/GBV-C RNA in serum (Pessoa et al, 1997). The assay employed target probes based on the relatively conserved sequence in the 5 untranslated region of the HGV/GB V-C genome. Preamplifier molecules and incorporation of isoC and isoG into the sequences common to bDNA assays were used to enhance the analytical sensitivity. The provisional limit of detection was 32,500 genome equivalents/ml based on dilutions of a 700-nucleotide synthetic HGV/GBV-C RNA transcript. The run-to-run variance of the assay was <15%. 

The obvious similarity between the purine bases of DNA and pteridines, especially between guanosine and pterins, has encouraged extensive studies of the synthesis and properties of pteridine-containing nucleoside and nucleotides. Synthetic methods have naturally built upon established methods of nucleic acid synthesis. The primary property of use in applications of these compounds to DNA chemistry is fluorescence, which is very much greater for pteridines than for purines. 

As the density of information derived from efforts to sequence, map and identify human genes increased, so did the demand for analytical tools capable of exploiting this information. DNA microarrays were developed in response to this demand. Southern(69) was the first to describe parallel, in situ ohgonucleotide synthesis as a means of generating oligonucleotide probe arrays on solid supports for highly parallel hybridization analysis. Southern s method uses standard nucleotide synthetic reactions to synthesize the oligonucleotides. The reactions are carried out in a movable chamber, which provides a physical barrier between the reaction chamber and the intended synthesis area. 

Most of the carbon that flows through nucleotide synthetic pathways goes into ribonucleotide triphosphates (rNTPs - ATP, CTP, GTP, and UTP). A relatively small fraction is diverted to the synthesis of deoxyribonucleoside triphosphates (dNTPs). rNTPs are synthesized in excess of dNTPs because most cells contain 5-10 times as much RNA as DNA and because rNTPs have multiple metabolic roles, whereas dNTPs are used only to make DNA. 

Despite structural diversity in the superactive enzymes of individual families, studies of PRPP and purine metabolism carried out both vivo and in cells cultured from affected hemizygous males support the idea that a common mechanism accounts for the association of PRPP synthetase superactivity with uric acid overproduction. Increased intracellular PRPP concentrations and rates of PRPP generation as well as increased rates of all PRPP-dependent purine nucleotide synthetic processes are constant accompaniments of enzyme superactivity. These findings suggest a scheme to explain the association of the enzyme defect with uric acid overproduction PRPP synthetase superactivity -> increased intracellular PRPP generation and concentration > increased rate of purine nucleotide synthesis excessive uric acid synthesis. 

Di- and trinucleotides may be used as units instead of the monomers. This convergent synthetic strategy simplifies the purification of products, since they are differentiated by a much higher jump in molecular mass and functionality from the educls than in monomer additions, and it raises the yield. We can illustrate the latter effect with an imaginary sequence of seven synthetic steps, c.g. nucleotide condensations, where the yield is 80% in each step. In a converging seven-step synthesis an octanucleotide would be obtained in 0.8 x 100 = 51% yield, compared with a 0.8 x 100 = 21% yield in a linear synthesis. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

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. 

LDH-x is one of the best characterized antigens and its amino acid sequence is known. A synthetic peptide based on a portion of the molecule has been shown to reduce fertUity in laboratory animals. The nucleotide sequence coding for human LDH-x has been defined and engineered into an expression vector system (121). 

It has been recrystd from H2O (fine needles) and is freely soluble in boiling H2O. Crysts also from H2O by addition of acetone. Purified by chromatography on Dowex 1 (in formate form), eluting with 0.25M formic acid. It was then adsorbed onto charcoal (which had been boiled for 15min with M HCI, washed free of chloride and dried at 100°), and recovered by stirring three times with isoamyl alcohol/H20 (1 9 v/v). The aqueous layer from the combined extracts was evaporated to dryness under reduced pressure, and the product was crystallised twice from hot H2O. [Morrison and Doherty Biochem J19 433 7967]. It has A-max 259nm (e 15,400) in H2O at pH 7.0. [Alberty et al. J Biol Chem 193 425 7957 Martell and Schwarzenbach Heh Chim Acta 39 653 7956]. The acridinium salt has m 208° [Baddiley and Todd J Chem Soc 648 1947 Pettit Synthetic Nucleotides, van Nostrand-Reinhold, NY, Vol 1 252 1972 NMR Sarma et al. J Am Chem Soc 96 7337 1974 Norton et al. J Am Chem Soc 98 1007 1976 IR of diNa salt Miles Biochem Biophys Acta 27 324 1958],... 

The protein-DNA interactions have been analyzed in detail at high resolution in the complex between the 434 repressor fragment and the ORl containing 20mer DNA. A pseudo-twofold symmetry axis relates the halves of this complex. The symmetry is not exact since the nucleotide sequence of the DNA is slightly different in each half (see Table 8.2). However, the interactions between one protein subunit and one half of the DNA are very similar to those between the second subunit and the other half of the DNA since most of the bases that interact with the protein are identical in both halves. Details of the interaction are very similar to those in the complex with the palindromic synthetic 14mer of DNA shown in Figures 8.14 and 8.15. The base pairs at one end of the DNA, 1-14, 2-13, etc. are called base pairs 1, 2, etc. 

DNA synthesizers operate on a principle similar to that of the Merrifield solid-phase peptide synthesizer (Section 26.8). In essence, a protected nucleotide is covalently bonded to a solid support, and one nucleotide at a time is added to the growing chain by the use of a coupling reagent. After the final nucleotide has been added, all the protecting groups are removed and the synthetic DNA is cleaved from the solid support. Five steps are needed ... 

FIG. 13 Synthetic DN A motifs for the construction of DNA framework Three- [75] (13) and four-arm (14) DNA junction [8] DNA double-crossover (DX) molecules 15,16 were used for initial studies of enzymatic oligomerization [79]. The DX motif 17, containing four cohesive ends of individual nucleotide sequence, was used for the construction of two-dimensional DNA crystals [80]. 

Synthetic nonhydrolyzable analogs of nucleoside triphosphates (Figure 33-13) allow investigators to distinguish the effects of nucleotides due to phosphoryl transfer from effects mediated by occupancy of allosteric nucleotide-binding sites on regulated enzymes. 

Synthetic analogs of purine and pyrimidine bases and their derivatives serve as anticancer dmgs either by inhibiting an enzyme of nucleotide biosynthesis or by being incorporated into DNA or RNA. 

The automated chemical synthesis of moderately long oligonucleotides (about 100 nucleotides) of precise sequence is now a routine laboratory procedure. Each synthetic cycle takes but a few minutes, so an entire molecule can be made by synthesizing relatively short segments that can then be ligated to one another. Oligonucleotides are now indispensable for DNA se-... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

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