摘要:

ABSTRACTUsing as a model the inhibition of β-glucuronidase expression in preimplantation embryos, we have compared injections of transgenes directing the synthesis of antisense RNA and antisense oligodeoxynucleotides to our previous results with cytoplasmic injections of antisense RNAs. Pronuclear injection of an antisense DNA construct containing 1.4 kb of coding region of β-glucuronidase fused to the mouse metallothionein I promoter results in transient inhibition of gene expression in preimplantation mouse embryos. Pronuclear injection of a smaller antisense DNA construct, overlapping the start codon, failed to inhibit gene expression. Injection of two 20-mer antisense oligodeoxynucleotides, one complementary to sequences including the initiation codon and the second complementary to exon 7 sequences of the β-glucuronidase gene, failed to inhibit gene expression. In addition, cultures of embryos in the presence of antisense oligodeoxynucleotides had no effect on gene activity. Using radiolabeled oligomers added to the culture medium, we found poor uptake of oligodeoxynucleotides by embry

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.1

年代:1991

数据来源: MAL

摘要:

ABSTRACTRecently, we described a new class of antisense oligonucleotides that can be used to direct the cleavage of mRNAs inXenopus laevisembryos by RNase H (Dagleet al., Nucleic Acids Res. 18, 4751–4757). In this study, we have examined several factors that determine the activity of these derivatives. In embryos, oligodeoxyribonucleotides were found to be rapidly degraded by a 3′ exonuclease. Modification of 3′-terminal phosphodiester linkages as phosphoramidates blocks this activity. The predominant sites of endonucleolytic cleavage within the embryo are localized close to the 5′ termini demonstrating the necessity of multiply modifying phosphodiester linkages at each end of the molecule. A stretch of at least six consecutive phosphodiester linkages is required to form an effective substrate forXenopusRNase H; mRNA degradation with an oligonucleotide containing fewer than six contiguous unmodified internucleoside linkages is greatly diminished. Injection of an anti-cyclin B oligonucleotide containing eight unmodified residues results in degradation of cyclin B mRNAs and subsequent inhibition of embryonic cell division. An oligonucleotide with the same sequence but containing four consecutive phosphodiesters has no observable effect on the cell cycle. This last observation suggests that, inXenopusembryos, hybridization alone has a limited role, if any, in oligonucleotide-mediated inhibition of gene exp

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.11

年代:1991

数据来源: MAL

摘要:

ABSTRACTUsing an antisense RNA approach to eliminate endogenous expression of the c-fosprotein, we have verified by nuclear run-on and transient expression assays that the Fos protein is a negative regulator of its own transcriptionin vivo. The negative autoregulation of the c-fospromoter by Fos was further confirmed by overexpression of an antisense-resistant c-fosexpressing vector. Antisense mapping of the c-fospromoter demonstrated that the serum responsive element (SRE) represents the major site for c-fossuppression only during the first hour, but that additional adjacent DNA sequences are required for suppression at later times. We propose that antisense inhibition of transcriptional repressors provides a useful method for analyzing the significance and mechanism of transcriptional repressionin vivo.

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.21

年代:1991

数据来源: MAL

摘要:

ABSTRACTRat adipocytes were treated with antisense dimethoxytrityl pentadecadeoxynucleotides, complementary to mRNA initiation codon regions for α and β isozymes of protein kinase C (PKC). This antisense treatment provoked 50–70% decreases in PKC and insulin-stimulated 2-deoxyglucose uptake, but did not inhibit insulin-stimulated diacylglycerol synthesis. Sense or nonsense oligodeoxynucleotides were without effect on PKC and 2-deoxyglucose uptake. These results suggest that: (i) PKC-α and PKC-β isozymes can be specifically downregulated in rat adipocytes by antisense oligodeoxynucleotides, and (ii) insulin-stimulated glucose transport require

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.35

年代:1991

数据来源: MAL

摘要:

ABSTRACTOligopyrimidines covalently linked to ellipticine derivatives form duplex and triplex structures with target single-stranded oligopurine sequences. They also bind to duplex DNA at homopurine–homopyrimidine sequences where they form local triple helices. Irradiation at wavelengths longer than 300 nm of the complex formed by an oligonucleotide–ellipticine conjugate with its target sequence induced (i) cleavage of the target at bases located in close proximity to the dye and (ii) cross-linking of the target sequence to the derivatized oligonucleotide. Both cross-linking and cleavage reactions decreased when temperature increased with a half-transition corresponding to the dissociation of the oligonucleotide-ellipticine conjugate from its target nucleic acid, demonstrating that the observed photochemical effects are dependent on hybrid formation. When the target was a double-stranded DNA, photochemical reactions were observed on both strands of the duplex. Photo-induced cross-linking was more efficient than cleavage when the target was single-stranded; the reverse was observed when the target was duplex

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.43

年代:1991

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.55

年代:1991

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.57

年代:1991

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.65

年代:1991

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1991.1.v

年代:1991

数据来源: MAL