Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.     

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

Summary Quantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.     

Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.     

Synthesis of d(GC) and d(CG) octamers containing alternating phosphorothioate linkages: effect of the phosphorothioate group on the B-Z transition

The synthesis of four oligonucleotides containing alternating phosphorothioate groups, (Rp)-and (Sp)-d[G(p(S)CpG)3p(S)C] and (Rp)- and (Sp)-d[C(p(S)GpC)p(S)G], by the phosphite approach is described. Silica gel to which 2'(3')-O-acetyluridine and 5'-succinyl groups were bound served as support for oligomer synthesis. The syntheses were carried out by dimer addition with presynthesized diastereomerically pure dinucleoside phosphorothioates as building blocks. The products were characterized by 31P NMR, nuclease P1 digestion, and oxidation to the corresponding all-phosphate-containing oligomers. The ability of each oligomer to adopt the Z conformation under high-salt conditions was screened for by circular dichroism spectroscopy. Both (Rp)-d[G(p(S)CpG)3p(S)C] and (Sp)-d[C(p(S)GpC)3p(S)G] are capable of forming Z-type structures at high NaCl concentrations. In the case of (Rp)-d[G(p(S)CpG)3p(S)C] where a phosphorothioate of the Rp configuration occurs 5' to a deoxycytidine residue, the B----Z transition is potentiated in comparison to the unmodified oligomer. (Sp)-d[G(p(S)CpG)3p(S)C] and (Rp)-d[C(p(S)GpC)3p(S)G] retain the B conformation even at high NaCl concentration.     

Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis.

M13 RF IV DNA where phosphorothioate groups are incorporated at restriction endonuclease Nci I recognition sites in the (-)strand is efficiently nicked by the action of this enzyme. Incubation of such nicked DNA with exonuclease III produces gapped DNA. The gap can be filled by reaction with deoxynucleoside triphosphates and DNA polymerase I. When this sequence of reactions is performed with DNA containing a mismatch oligonucleotide primer in the (-)-strand mutational frequencies of 70-90% can be obtained upon transformation. The general nature of this methodology has been further shown to be applicable to other restriction enzymes such as Hind II, Pst I and Fsp I. The mutational frequency obtained using these enzymes is between 40-80% mainly because of less efficient nicking and gapping. Studies on inhibition of Nci I cleavage show that in addition to a phosphorothioate group at the position of cleavage an additional group in the 5'-neighbouring position is necessary for complete inhibition.