We propose to use a newly developed procedure to analyze the in vivo DNA and chromatin organization of genes. Circular molecules of DNA labeled with 32p at a single restriction site are injected into oocytes and into fertilized egges of Xenopus laevis. The molecules can be retrieved and analyzed after they are assembled into chromatin, or after DNA replication in the case of the Xenopus empbryo. We find that Xenopus genes thus injected acquire an active chromatin conformation. We shall analyze the homologous, injected Xenopus histone genes and 5 S RNA genes under various experimental conditions to determine the molecular structure of the actively transcribing form and the inactive form of these eucaryotic genes. In a second, related project, we will use tagged hemimethylated MT3 DNA to determine the timing and pattern of DNA methylation in the Xenopus embryo. The injected DNA will be retrieved and sequenced by the Maxam and Gilbert technique to locate the 5-methyl-cytosine (5mC) residues on both the (+) and (-) DNA strands within a 600 bp region of M13 DNA. The methylation pattern will be analyzed after one and two rounds of DNA replication during the physiologically synchronous cleavage events which occur after fertilization of the eggs by sperm. These experiments will establish 1) whether DNA demethylation can occur on some 5mC containing DNA sequences, 2) which 5mC-containing DNA sequences are propagated to the complementary DNA strand and 3) when, during the cell cyle (G1, S, G2), are these particular 5mC sequences propagated. Once the general rules of inheritance of DNA methylation are elucidated, we shall examine the effect of DNA methylation on the chromatin organization and gene expression of the injected Xenopus histone genes and 5 S RNA genes.
| Kmiec, E B; Ryoji, M; Worcel, A (1986) Gyration is required for 5S RNA transcription from a chromatin template. Proc Natl Acad Sci U S A 83:1305-9 |
