RNA localization in Xenopus oocytes.
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Biomedical subjects
J Yisraeli
Publications and source records attributed to J Yisraeli.
Dynamics of demethylation and activation of the alpha-actin gene in myoblasts.
Transient transfection into L8 myoblasts has been used to study the rat alpha-actin gene promoter. Demodification of specific sites occurs in two stages, with a hemimethylated intermediate formed within a few hours after entry of the alpha-actin gene construct into the cell. The removal of the methyl moiety from the complementary strand takes place after a delay of at least 48 hr, and both events are actively carried out in the absence of DNA replication. By assaying gene activity during the course of the transfection, it was possible to demonstrate that demethylation of both strands at the critical CpG loci is essential to activate transcription. Genetic analysis revealed the existence of cis-acting elements required for demethylation. The recognition of these sites early in the differentiation process probably leads to the demodification events required to make the gene accessible to its transcription factors.
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Localization of mRNA and axis formation during Xenopus embryogenesis.
This paper summarizes our recent work concerned with the developmental polarity of the frog egg and the patterning of the embryonic body plan. In two separate projects, we are studying genes involved in setting up basic embryonic axes. One of these genes, Vg1, codes for a maternal mRNA that is localized in the frog egg. The Vg1 gene is used in studies on the induction of mesoderm and as a probe to understand how the polarity of an egg is established. A second gene, Xhox3, contains a homeodomain and is differentially expressed in the axial mesoderm. Our studies suggest that this homeobox gene is critically involved in setting up different positional values along the anteroposterior axis.
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Effect of in vitro DNA methylation on beta-globin gene expression.
When the human beta-globin gene was methylated at every cytosine residue and was inserted into mouse fibroblasts by DNA-mediated gene transfer, the transcription of the gene was strongly inhibited. This methylation also prevented expression and induction of the gene in mouse erythroleukemia cells. By using partially methylated hybrid molecules, it was shown that methylation-sensitive negative regulatory elements are located in both the 5' and 3' ends of the beta-globin gene but not in the 90-base-pair region usually associated with promoter activity. To further investigate the role of DNA methylation in the regulation of the beta-globin gene, 50-base-pair poly(dG-dC) tracts were introduced into various sites in a mouse-human hybrid gene, and these inserts were methylated by means of the Hha I methylase. Heavy methylation of these artificially added sites had no effect on either transcription initiation or elongation, suggesting that DNA modification operates through fixed endogenous sites in the gene domain.
Animals↗
Muscle-specific activation of a methylated chimeric actin gene.
To understand how DNA methylation affects tissue-specific activation of genes, we have transfected in vitro methylated alpha-actin (skeletal) constructs into fibroblasts, which do not produce endogenous alpha-actin, and into a myogenic line, which is inducible for alpha-actin expression. Although methylation significantly inhibits the expression of these constructs in fibroblasts, it does not in myoblasts. The methylation pattern of the introduced methylated genes reveals specific demethylations in the transfected molecules in myoblasts but not in fibroblasts, and it precisely mimics the methylation pattern found in myoblasts in vivo.
Actins↗
Effect of regional DNA methylation on gene expression.
The effect of DNA methylation on the transcriptional activity of the hamster adenine phosphoribosyltransferase (aprt) and the herpes thymidine kinase (tk) genes has been investigated. By using M13 constructs containing these gene sequences, specific segments of each gene were methylated in vitro by restriction fragment primer-directed second-strand synthesis using the substrate 2'-deoxy-5-methyl-cytidine triphosphate (dmCTP). These hybrid-methylated molecules were inserted into mouse Ltk- cells by DNA-mediated cotransfer. In all cases, the integrated sequences retained the in vitro-directed methylation pattern. The aprt gene was inhibited by CpG methylation in the 5' region but was unaffected by methylation at the 3' end or in adjacent M13 sequences. In contrast to this, DNA methylation in both the 5' promoter region and the 3' structural region of the tk gene had a strong inhibitory effect. This suggests that this modification may affect transcription by mechanisms that do not involve the direct alteration of recognition sequences for RNA polymerase.
Adenine Phosphoribosyltransferase↗
Variations in DNA methylation during mouse cell differentiation in vivo and in vitro.
Mouse teratocarcinoma cells induced to differentiate in vitro undergo a massive (30%) demethylation of DNA. A similar undermethylation is also observed in the mouse extraembryonic membranes, the yolk sac and placenta. In both cases, the decrease in methyl moieties occurs at a large number of CpG sites spread out over the entire genome, as indicated by a restriction enzyme analysis of several mouse genes including dhfr, beta-major globin, and the H-2K gene family. In contrast to this, the embryo itself appears to undergo methylation de novo during early stages of embryogenesis. Thus, as opposed to somatic cells, events during early mouse development are associated with wide variations in the level of DNA methylation. Although these changes in DNA methylation seem to be an integral part of the differentiation process, its relation to specific gene expression is still unclear.
Animals↗