Association of transcriptionally active vitellogenin II gene with the nuclear matrix of chicken liver

Supercoiled DNA loops linked to the nuclear matrix can be progressively cleaved with deoxyribonuclease I. The DNA which remains associated with the nuclear matrix can be purified and analysed for vitellogenin II sequence content by dot blot hybridization. Using this technique we show that vitellogenin II gene sequences are selectively associated with the nuclear matrix of liver but not with oviduct of laying hens. Following primary stimulation in immature chicks of vitellogenin synthesis with estradiol, the association of the gene with the nuclear matrix precedes vitellogenin mRNA synthesis. After 15 days when the level of vitellogenin mRNA has returned to zero, the gene is no longer preferentially associated with the nuclear matrix. At this time a second stimulation with estradiol results in a reassociation of the vitellogenin II gene with the nuclear matrix. In addition to the structural gene, both the 3' and 5' end flanking regions (1.5-2 kb) also bind to the nuclear matrix. However, beyond the limit of 1.5-2 kb upstream from the 5' end of the gene, there is no preferential binding of DNA to the nuclear matrix.     

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.     

Recognition site of nuclear factor I, a sequence-specific DNA-binding protein from HeLa cells that stimulates adenovirus DNA replication.

Nuclear factor I is a 47-kd protein, isolated from nuclei of HeLa cells, that binds specifically to the inverted terminal repeat of the adenovirus (Ad) DNA and enhances Ad DNA replication in vitro. We have studied the DNA sequence specificity of nuclear factor I binding using cloned terminal fragments of the Ad2 genome and a set of deletion mutants. Binding of nuclear factor I protects nucleotides 19-42 of Ad2 DNA against DNase I digestion. Filter binding assays show that deletion of the first 23 nucleotides does not impair binding while a deletion of 24 nucleotides reduces binding severely. However, binding studies on Ad12 DNA indicate that nucleotide 24 can be mutated. Fragments containing the first 40 bp are bound normally while the first 38 bp are insufficient to sustain binding. Taken together, these results indicate that the minimal recognition site of nuclear factor I contains 15 or 16 nucleotides, located from nucleotide 25 to nucleotide 39 or 40 of the Ad2 DNA. This site contains two of the four conserved nucleotide sequences in this region. Sequences flanking the minimal recognition site may reduce the binding affinity of nuclear factor I. In accordance with these binding studies, DNA replication of a fragment that carries the sequence of the terminal 40 nucleotides of Ad2 at one molecular end is enhanced by nuclear factor I in an in vitro replication system.