Silencing and trans-activation of the mouse IL-2 gene in <i>Xenopus</i> oocytes by proteins from resting and mitogen-induced primary T-lymphocytes

The Xenopus oocyte system was used to test functionally, putative trans‐active elements involved in the transcriptional control of the mouse interleukin‐2 (IL‐2) gene in resting and mitogen‐induced primary T‐lymphocytes. The IL‐2 gene injected into the oocyte is active over a wide range of DNA concentrations. This basal activity is silenced by the addition of protein extracts from G0‐arrested spleen cells. Extracts from 8 h‐stimulated spleen cells do not silence but moderately increase transcription over basal level. When IL‐2 transcription is silenced first by an injection of extract from resting spleen cells, the addition of proteins from stimulated cells results in a strong increase in transcription (derepression). Use of proteins from purified splenic T‐lymphocytes shows that both silencer(s) and activator(s) are contributed by these cells. Extracts from control tissues have neither a silencing nor stimulatory effect. None of the proteins tested affects the activities of co‐injected control genes. Injections with IL‐2 promoter mutants indicate that the main target sequence of the silencing and activating factors is a purine region (Pu‐box) lying between positions −261 and −292 upstream of the IL‐2 gene. Bandshift assays show differential binding of the Pu‐box with proteins from resting or activated T‐cells. Previous Article Next Articl

Inhibition of <i>Xhox1A</i> gene expression in <i>Xenopus</i> embryos by antisense RNA produced from an expression vector read by RNA polymerase III

Antisense inhibition of gene expression during Xenopus development was obtained by injecting, into the zygote, an expression vector carrying the adenovirus VAI gene read by RNA polymerase III. This vector yields high levels of antisense RNA in most embryonic cells between mid-blastula transition and tailbud stage. As a target we chose the Xenopus homeobox gene Xhox1A. A 26 bp long oligonucleotide, including the initiation codon of this gene, was inserted in opposite polarity into the vector. Antisense treatment reduces Xhox1A mRNA in embryos up to stage 22 and Xhox1A protein expression up to stage 30. Half of the antisense-treated embryos develop a characteristic phenotype with disorganized somites in the anterior trunk and delayed development of the intestinal tract

Oocyte shuttle, a recombinant protein transporting donor DNA into the Xenopus oocyte in situ

The newly developed oocyte shuttle protein contains a streptavidin moiety that tightly binds biotinylated DNA. Injected intravenously into adult Xenopus females, the protein-DNA complex is rapidly transported through the bloodstream and, within the ovary, the vitellogenin ligand present in the protein binds to the receptors at the surface of the oocytes. The bound complex is internalized and translocates into the oocyte nucleus thanks to an SV40 nuclear localization signal, enhanced by an adjacent casein kinase phosphorylation site. Functioning of the shuttle protein is documented by transporting DNA molecules that, upon intramolecular homologous recombination within the oocyte nucleus, express easily traceable markers such as green fluorescence or tetracycline resistance

A repression-derepression mechanism regulating the transcription of human immunodeficiency virus type 1 in primary T cells.

BACKGROUND: Despite some controversy regarding the preferential infection and replication of human immunodeficiency virus type 1 (HIV-1), it appears that primary T lymphocytes, in their quiescent state, are nonpermissive for viral expression and propagation. Massive activation of viral gene expression occurs only when the host lymphocyte is activated. These observations prompted us to investigate the transcriptional regulation of HIV-1 in resting or activated T cells that were isolated from cord blood or adult peripheral blood. MATERIALS AND METHODS: To this end, we employed cellular purification and phenotyping techniques, in vitro protein-DNA binding studies, functional transactivation assays using proteins isolated from cord blood or adult peripheral blood T lymphocytes, and transfection experiments in primary T cells. RESULTS: We showed that transcription from the HIV-1 long terminal repeat is repressed in resting naive T lymphocytes; whereas, mitogenically stimulated CD4+ cells form an activator that derepresses transcription. Negative and positive regulation act through a repressor-activator target sequence (RATS), which shares homology with the interleukin-2 (IL-2) purine-rich response element, through the adjacent binding site of the nuclear factor of activated T cells (NFAT), and weakly, through the KB region. CONCLUSIONS: This regulation exerted by cellular transcription factors can account for several important features of HIV-1 expression in primary CD4+ cells. Tight repression in resting naive T helper cells may be a main cause of viral latency and transcriptional activation accounts for massive viral production in activated T lymphocytes