Characterization and modulation of immune stimulation by modified oligonucleotides.

Single-stranded oligodeoxynucleotides (ODNs) were tested for their ability to stimulate NK cells isolated from murine spleens to lyse target cells. Various sequences were evaluated, some of which have been shown previously to exhibit pharmacologic activity in murine model systems. It was confirmed that the CpG motif was stimulatory only in specific sequence contexts, and we found that phosphorothioate backbones were, in general, less stimulatory than phosphodiester backbones. In addition, this stimulation could be reduced by methylating the cytosine of the CpG and eliminated by modifying all of the cytosines contained in an ODN with methyl, bromo, or iodo modifications to the 5 position of the cytosine ring. These results were compared with the ability of a subset of these ODN sequences to stimulate B cell proliferation in vitro. In this comparison, phosphorothioate backbones were found to be required, and the context of the CpG motif was found to be less critical for activation. Finally, one of the most potent ODNs was shown to activate NK and B lymphocytes when administered in vivo.

Selective inhibition of A-Raf and C-Raf mRNA expression by antisense oligodeoxynucleotides in rat vascular smooth muscle cells: role of A-Raf and C-Raf in serum-induced proliferation.

Raf kinases, cytoplasmic serine/threonine protein kinases, have been proposed as important participants in mitogen-induced signal transduction. However, the precise role that Raf kinase isozymes play in cellular responses such as proliferation has not been resolved. The present study investigates the ability of antisense phosphorothioate oligodeoxynucleotides (ODNs), targeted against rat C-Raf and A-Raf kinases, to reduce gene expression and proliferation of cultured rat A10 smooth muscle cells (SMCs). Exposure of A10 cells to ISIS 11061, an active C-Raf antisense ODN, resulted in a potent, dose-dependent inhibition (IC50 = 55 nM) of C-Raf mRNA and protein expression. This inhibition was completely dependent on ODN sequence because the incorporation of increasing numbers of mismatches (up to six) into the sequence resulted in sequential loss of potency. Similarly, a dose-dependent reduction (IC50 = 125 nM) in A-Raf gene expression was observed after treatment of cells with the active A-Raf ODN, ISIS 9069, whereas two scrambled controls were without effect. These results demonstrate that ISIS 11061 and ISIS 9069 reduced gene expression in a sequence-specific and isozyme-specific manner. Moreover, administration of ISIS 11061 and ISIS 9069 to rat SMCs resulted in a significant and potent diminution of serum-induced proliferation with corresponding IC50 values of 216 and 273 nM, respectively. Taken together, these results indicate that A-Raf and C-Raf kinases play an important role in regulating vascular SMC proliferation and that antisense-mediated inhibition of Raf kinase activity may serve as a therapeutic modality in the treatment of vascular proliferative disorders.

Multiple portions of a small region of the Drosophila transformer gene are required for efficient in vivo sex-specific regulated RNA splicing and in vitro sex-lethal binding.

The transformer gene of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing in transgenic animals. This entire region appears to be necessary for regulation and for efficient Sex-lethal binding. Natural splice sites containing partial homology to transformer do not show regulation. Mutations which replace the 16 nucleotides surrounding the branch point or alter single nucleotides near the splice site eliminate or reduce regulation without eliminating splicing. Mutations which reduce or eliminate regulation in vivo reduce binding to Sex-lethal in vitro, consistent with the hypothesis that these mutations bring about their effects by altering Sex-lethal binding rather than by altering binding sites for additional non-Sex-lethal factors.

Molecular genetics of transformer, a genetic switch controlling sexual differentiation in Drosophila.

The transformer gene is one of a set of regulatory genes that form the hierarchy controlling all aspects of somatic sexual differentiation in Drosophila melanogaster. The gene transformer occupies an intermediate position in this hierarchy. Analysis of this gene has allowed us to determine the mechanism by which it is regulated in a sex-specific manner and to examine the way in which the regulatory hierarchy is organized. The female-specific expression of the tra gene, previously inferred from genetic observations, is based on sex-specific alternative splicing of tra pre-mRNA and is not the result of sex-specific transcriptional activation. The female-specific RNA produced by this alternative splicing is the functional mediator of tra activity. Multiple genetic, molecular, and transformation experiments show that female-specific activation of genes or gene products occurs in the order Sex lethal greater than transformer greater than transformer-2 greater than doublesex greater than or equal to intersex greater than female differentiation. The results do not distinguish the level at which transformer might regulate the downstream gene transformer-2. Neither transformer nor any of the down-stream genes feedback on, or participate in, alternative splicing of transformer RNA. The mechanism by which Sex lethal regulates transformer splicing appears to be a repression of the use of one of a pair of splice acceptor sites.

Ectopic expression of the female transformer gene product leads to female differentiation of chromosomally male Drosophila.

The transformer (tra) gene of Drosophila is necessary for all aspects of female somatic sexual differentiation. tra uses a single set of precursor RNAs to produce female- and non-sex-specific RNAs by alternative splicing. Ectopic expression of the female-specific RNA causes chromosomal males to develop as females, indicative of a linear pathway of regulated genes controlling sex. Genetic and molecular tests with this ectopically expressed gene are consistent with the following order of gene action: X chromosome to autosome ratio----Sex lethal----transformer----transformer-2----doublesex----intersex--- - terminal differentiation. Expression of the female-specific tra RNA in tra mutants is sufficient to lead to female differentiation. Expression of the non-sex-specific tra RNA in tra mutants is not sufficient to lead to female differentiation. The tra female-specific activity is not required for female-specific splicing of the tra precursor RNAs.

Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene.

The transformer (tra) gene regulates female somatic sexual differentiation and has no known function in males. It gives rise to two sizes of RNA, one non-sex-specific and one female-specific. These two RNAs are shown to be present throughout the life cycle, and related by the use of alternative first intron splice acceptor sites. The non-sex-specific RNA has a 73 base first intron, while that in the female-specific RNA is 248 bases. The non-sex-specific RNA has no long open reading frame, while the female-specific RNA has a single long open reading frame beginning at the first AUG. Substitution of a heat shock promoter for the tra promoter still leads to female-specific differentiation of otherwise tra-females. We suggest a mechanism by which Sex-lethal controls itself and tra.

Higher plant chloroplasts: Evidence that all the chlorophyll exists as chlorophyll-protein complexes.

By using the polyacrylamide gel electrophoresis system described in this report, it was possible to fractionate all the photosynthetic pigments of maize (Zea mays L.) thylakoids into chlorophyll-protein complexes with negligible formation of free or detergent-complexed chlorophyll. Identical sodium dodecyl sulfate extracts of thylakoids have previously resulted in up to 50% of the chlorophyll migrating as free chlorophyll after electrophoresis. The major difference from previous gel electrophoresis systems is the replacement of sodium dodecyl sulfate in the electrophoresis buffer by Deriphat 160 (disodium N-lauryl-beta-iminodipropionate), a zwitterionic detergent. The results suggest that: (i) no significant amount of free chlorophyll exists in the chloroplast thylakoid membranes in vivo, and (ii) most of the free pigment seen previously on gels was generated during the electrophoresis and was not a result of the solubilization technique. Additionally, the new chlorophyll-protein complexes resolved appear to have different characteristics (pigment content and size) that those observed in former systems.