DNaseI hypersensitive site mapping of the F rubripes αβ-globin gene domain.

(A) Features of the F rubripes αβ-globin gene domain and strategy for DNaseI hypersensitive site mapping. Arrows indicate the transcriptional direction of the genes. Restriction sites used to cut genomic F rubripes DNA are indicated: A,SacI; E, EcoRI; S, SphI; X,XhoI. The EcoRI sites were used to isolate the fragment for transgenesis. Probes for Southern hybridization were generated by PCR amplification and are labeled 1 to 7. Repetitive sequences are denoted by gray bars: T indicates homology with a human telomeric sequence (Z9627522); ?, unknown repetitive element with homology to yeast tRNA-met (AL121 795,); R, areas with multiple BLAST hits in the Fugu genome; R1, homology with reverse transcriptase/rex1 transposon.23 The positions of strong erythroid-specific hypersensitive sites are indicated by black arrowheads; weak hypersensitive sites are indicated by open arrowheads. (B-C) Examples of Southern blot analysis of DNaseI hypersensitive sites in F rubripes chromatin. Nuclei were isolated from the tissues indicated and treated with increasing amounts of DNaseI. DNA was purified, digested with the appropriate restriction enzymes, and subjected to Southern blotting. In panel B, the DNA was digested withSphI, and the blot was hybridized with probe 5. Strong hypersensitive sites coinciding with the promoters of the α3- and β-globin genes are indicated by arrows. Arrowheads indicate weaker sites. In panel C, the DNA was digested with SacI, and the blot was hybridized with probe 5. No hypersensitive sites are observed in blood, kidney, or liver (not shown).