Phosphorothioate Analogues of Nucleotides—Tools for the Investigation of Biochemical Processes

Eckstein, Fritz

doi:10.1002/anie.198304233

Cited by 218 publications

(129 citation statements)

References 194 publications

Supporting

Mentioning

125

Contrasting

Order By: Relevance

“…Mutagenesis was directed to UAS1 by employing a modification of the misincorporation procedure that has been described previously (6,25). The method we employed localized the misincorporation of single a-thiophosphate deoxyribonucleotides to a 100-bp region spanning UAS1.…”

Section: Methodsmentioning

confidence: 99%

“…The fragments were inserted into a pBL5Int backbone VOL. 6,1986 -IL-f-extending from the SmaI site at -312 to the BamHI site at the CYCI-lacZ fusion. The resulting plasmids contain approximately 1,100 bp of CYCI DNA upstream of the CYCIlacZ fusion, including either the wild-type UAS1 (pBLAUAS2Int) or a UAS1 base substitution mutation, and are deleted for UAS2 and for the 2pum origin.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

Lalonde¹,

Arcangioli²,

Guarente³

1986

Mol. Cell. Biol.

View full text Add to dashboard Cite

Several site-directed mutagenesis regimens were used to generate single-and multiple-base substitutions in the upstream activation site UAS1 of the Saccharomyces cerevisiae CYCI gene. Mutations resulting in large reductions in activity of the site lie in two distinct regions. Six single-base changes in a region A, between -288 and -285, all resulted in a 15-fold reduction in activity. Synthetic sites built up solely of multimers of the -289 to -285 sequence ACCGA behaved as carbon catabolite-sensitive UASs. In addition, substitution mutations in a second region, at nucleotides -266 and -265, virtually eliminated UAS1 activity. These mutations abolished the binding of a heme-dependent protein factor in vitro. Thus, UAS1 contains two essential regions both of which are required for its activity.Upstream activation sites (UASs) are cis-acting elements found upstream of TATA boxes that are required for efficient transcription of all Saccharomyces cerevisiae genes analyzed to date (for a review, see reference 10). UASs constitute a primary route by which gene-specific regulation is conferred (5, 12-14, 21, 26).The sites appear to have several features in common with analogous sites, termed enhancers, identified in higher eucaryotes (reviewed in references 4, 9, 19). For example, the yeast sites function at variable distances from the TATA box or when their orientation with respect to the TATA box is inverted (11). The activity of UASs and enhancers very likely depends on trans-acting proteins that bind to the sites. In mammalian systems both the mouse mammary tumor virus (MTV) enhancer and the simian virus 40 distal regulatory region consist of composites of short repeated elements. The MTV element is an octamer to which the glucocorticoid receptor binds (28), and the simian virus 40 element is a hexamer to which Spl binds (7). In yeasts, galactose inducibility can be mediated by a 17-base-pair (bp) dyad to which the GAL4 protein binds (3,8). Further, a hexamer has been identified genetically as the UAS mediating general control of many amino acid biosynthetic genes (5, 15, 16). Binding of the product of the GCN4 locus to this sequence will occur in vitro but is dependent on sequences that flank the hexamer (18).Regulation of the CYCI gene which encodes iso-1-cytochrome c is governed by two distinct UAS sites, UAS1 located at -275 and UAS2 located at -225 ( Fig. 1) (2). By fusing these sites separately to the LEU2 TATA box, we showed that their activity requires heme: both sites display a very low level of activity under heme-deficient conditions and are induced about 200-fold by the addition of heme to growth media (12, 13). Under heme-sufficient conditions, the sites are further derepressed by shifting from glucose medium to one containing a nonfermentable carbon source, e.g., lactate. UAS1 may also be derepressed by the addition of the heme analog deuteroporphyrin IX to glucose medium. Each UAS site can function independently of the other, and the effect of both sites in the intact promoter is roughly additive. Al...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

Lalonde¹,

Arcangioli²,

Guarente³

1986

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…The configuration of the phosphorothioate linkage of the 2Ј,5Ј phosphorothioate oligoadenylates is also determined by selective enzyme digestion using snake venom phosphodiesterase that degrades the Rp-phosphorothioate linkage but not the Sp-phosphorothioate linkage. 15,17,21,23,29 The steric course of the enzymatic reactions of oligonucleotides and phosphate derivatives has been reported to be markedly changed by creating a chiral center by replacing a nonbridging oxygen of a phosphodiester bond with a sulfur. [15][16][17] We also confirmed the assignment of the configuration of the 2Ј,5Ј phosphorothioate oligoadenylates by a comparison of their retention times of the reverse-phase HPLC.…”

Section: And Nmr Studies Of Phosphorothioate Analogues Of 2-5amentioning

confidence: 99%

“…15,17,21,23,29 The steric course of the enzymatic reactions of oligonucleotides and phosphate derivatives has been reported to be markedly changed by creating a chiral center by replacing a nonbridging oxygen of a phosphodiester bond with a sulfur. [15][16][17] We also confirmed the assignment of the configuration of the 2Ј,5Ј phosphorothioate oligoadenylates by a comparison of their retention times of the reverse-phase HPLC. 20,21 The 2Ј,5Ј Rp-phosphorothioate oligoadenylates have been reported to elute faster on a reverse-phase HPLC than the corresponding 2Ј,5Ј Sp-phosphorothioate oligoadenylates.…”

Section: And Nmr Studies Of Phosphorothioate Analogues Of 2-5amentioning

confidence: 99%

“…[7][8][9] Among the many synthesized 2-5A congeners, the phosphorothioate analogues of 2-5A are of special interest because they have enhanced stability against cellular nuclease and strong biological activity like that of native 2-5A. 10 -14 The phosphorothioate linkage has a chiral center at the phosphorous atom, which regulates the stereochemical course of the enzymatic process [15][16][17] such as binding to and activation of RNase L. 11,14 Enzymatically, 2Ј,5Ј phosphorothioate oligoadenylates with the Rp configuration are produced from Sp-ATP␣S by 2-5A synthetase. 12,13 2Ј,5Ј Phosphorothioate triadenylates with all possible thiophosphoryl configurations have been chemically prepared, 18 and their biological activities have been evaluated.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Conformational properties of 2′,5′ linked Rp‐ and Sp‐phosphorothioate oligoadenylates studied by circular dichroism and NMR

et al. 2002

View full text Add to dashboard Cite

2Ј,5Ј-Linked oligoadenylates (2-5A) are involved in the antiviral action of interferon. The 2-5A binds and activates 2-5A dependent RNase (RNase L), which degrades viral mRNA, resulting in the inhibition of protein synthesis. 2Ј,5Ј-Linked phosphorothioate oligoadenylates with an Rp configuration bind to and activate the RNase L. On the other hand, 2Ј,5Ј phosphorothioate oligoadenylate with an Sp configuration weakly binds to the RNase L and is devoid of the RNase L activation ability. Comparative circular dichroism (CD) and NMR studies are carried out to characterize the difference in properties between the two configurations of the 2Ј,5Ј phosphorothioate oligoadenylates. 2Ј,5Ј Rp-Phosphorothioate oligoadenylates showed CD spectra similar to those of the corresponding native 2Ј,5Ј oligoadenylates, while the 2Ј,5Ј Sp-phosphorothioate oligoadenylates exhibited a weaker CD band compared to the former two, indicating the weaker base-stacking interaction of the 2Ј,5Ј Sp-phosphorothioate oligoadenylates. The temperature-dependent change in the CD revealed that 2Ј,5Ј phosphorothioate oligoadenylates showed larger ⌬H°and ⌬S°values for the thermal transition of the conformation than the corresponding native 2Ј,5Ј oligoadenylates. The NMR spectral assignment was accomplished by several NMR measuring techniques. The 2Ј-H of the ribose ring linked to the 2Ј,5Ј Sp-phosphorothioate showed a higher field chemical shift of the proton NMR than that linked to the corresponding 2Ј,5Ј Rp-phosphorothioate. 2Ј,5Ј Rp-and Sp-phosphorothioate oligoadenylates possess a sugar conformation similar to that of the corresponding native 2Ј,5Ј oligoadenylates.

show abstract

Antisense oligonucleotides as therapeutic agents

1999

View full text Add to dashboard Cite

Antisense oligonucleotides can block the expression of specific target genes involved in the development of human diseases. Therapeutic applications of antisense techniques are currently under investigation in many different fields. The use of antisense molecules to modify gene expression is variable in its efficacy and reliability, raising objections about their use as therapeutic agents. However, preliminary results of several clinical studies demonstrated the safety and to some extent the efficacy of antisense oligodeoxynucleotides (ODNs) in patients with malignant diseases. Clinical response was observed in some patients suffering from ovarian cancer who were treated with antisense targeted against the gene encoding for the protein kinase C-alpha. Some hematological diseases treated with antisense oligos targeted against the bcr/abl and the bcl2 mRNAs have shown promising clinical response. Antisense therapy has been useful in the treatment of cardiovascular disorders such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy. Antisense oligonucleotides also have shown promise as antiviral agents. Several investigators are performing trials with oligonucleotides targeted against the human immunodeficiency virus-1 (HIV-1) and hepatitis viruses. Phosphorothioate ODNs now have reached phase I and II in clinical trials for the treatment of cancer and viral infections, so far demonstrating an acceptable safety and pharmacokinetic profile for continuing their development. The new drug Vitravene, based on a phosphorothioate oligonucleotide designed to inhibit the human cytomegalovirus (CMV), promises that some substantial successes can be reached with the antisense technique.

show abstract

Phosphorothioate Analogues of Nucleotides—Tools for the Investigation of Biochemical Processes

Cited by 218 publications

References 194 publications

A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

Conformational properties of 2′,5′ linked Rp‐ and Sp‐phosphorothioate oligoadenylates studied by circular dichroism and NMR

Antisense oligonucleotides as therapeutic agents

Contact Info

Product

Resources

About