Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enhancer of the human neurotropic papovavirus JC virus (JCV) restricts viral transcription to glial cells. We utilized the tissue specificity of the JCV enhancer as a tool to investigate the function of human immunodeficiency virus (HIV) Tat in transcriptional activation. The reporter plasmid pJCTAR-CAT was constructed by inserting the HIV type 1 Tat-responsive element, TAR, between the JCV promoter and the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of pJCTAR-CAT and pSV-Tat, an expression vector for Tat, resulted in a 50-fold increase in JCV promoter activity in cells nonpermissive for JCV expression. Both the 98-bp JCV enhancer and the HIV TAR sequences were required for transactivation of pJCTAR-CAT in nonpermissive cells. The transactivation by Tat occurred at the level of transcription, as the increase in CAT activity paralleled an increase in the steady-state levels of CAT mRNA in S1 nuclease and nuclear run-on analyses. In the presence of Tat, the JCV enhancer is functional in cells normally nonpermissive for JCV expression; therefore, our results provide unique evidence that HIV type 1 Tat may regulate the activity of specific transcription factors.
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PMID:Human immunodeficiency virus Tat transactivation: induction of a tissue-specific enhancer in a nonpermissive cell line. 165 61

Oligonucleotide analogues 15-20 nucleotides in length have been prepared, whose sequences are complementary to nucleotides in the upper hairpin of HIV TAR RNA. These alternating oligonucleoside methylphosphonates, mr-AOMPs, contain 2'-O-methylribonucleosides and alternating methylphosphonate and phosphodiester internucleotide linkages. The methylphosphonate and phosphodiester linkages of these oligomers are highly resistant to hydrolysis by exonuclease activity found in mammalian serum and to endonucleases, such as S1 nuclease. The oligomers were prepared using automated phosphoramidite chemistry and terminate with a 5'-phosphate group, which provides an affinity handle for purification by strong anion exchange HPLC. A 15-mer mr-AOMP, 1676, that is complementary to the 5'-side of the TAR RNA hairpin, including the 3-base bulge and 6-base loop region, forms a 1:1 duplex with a complementary RNA 18-mer, mini-TAR RNA. The T(m) of this duplex is 71 degrees C, which is similar to that of the duplex formed by the corresponding all phosphodiester 15-mer. Introduction of two mismatched bases reduces the T(m) by 17 degrees C. The apparent dissociation constant, K(d), for the 1676/mini-TAR RNA duplex as determined by an electrophoretic mobility shift assay at 37 degrees C is 0.3 nM. Oligomer 1676 also binds tightly to the full length TAR RNA target under physiological conditions (K(d) = 20 nM), whereas no binding was observed by the mismatched oligomer. A 19-mer that is complementary to the entire upper hairpin also binds to TAR RNA with a K(d) that is similar to that of 1676, a result that suggests only part of the oligomer binds. When two of the methylphosphonate linkages in the region complementary to the 6-base loop are replaced with phosphodiester linkages, the K(d) is reduced by approximately a factor of 10. This result suggests that interactions between TAR RNA and the oligomer occur initially with nucleotides in the 6-base loop, and that these interactions are sensitive to presence and possibly the chirality of the methylphosphonate linkages in the oligomer. The high affinities of mr-AOMPs for TAR RNA and their resistance to nuclease hydrolysis suggests their potential utility as antisense agents in cell culture.
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PMID:Syntheses of alternating oligo-2'-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA. 1056 19