Multiple interactions between nuclear proteins of Zea mays and the promoter of the Shrunken gene

Summary Nuclear proteins were extracted from isolated nuclei of immature maize kernels. The promoter region (1.5 kb) of the Shrunken gene, which is highly transcribed in the developing endosperm of the kernel, was scanned for protein-DNA interactions. Several promoter fragments showed protein-DNA complex formation in gel retardation experiments. Two different nucleo-protein complexes (MNP1 and MNP2) have been distinguished in competition and DNase I footprinting experiments. Both nuclear DNA-binding activities are able to recognize multiple sites distributed over a 1.5 kb upstream region of the Shrunken gene. Some of the binding sites established in the in vitro reconstitution experiments are located near to DNase I hypersensitive sites found in the promoter of the Shrunken gene (Frommer and Starlinger 1988).     

Binding of the estrogen receptor to DNA. The role of waters.

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.     

The protein factor which binds to the upstream activating sequence of Saccharomyces cerevisiae ENO1 gene.

Using a gel retardation assay it was shown that the 87 bp DNA fragment (UAS87) containing the upstream activating sequence (UAS) of S. cerevisiae EN01 gene and a nuclear extract gave rise to three migration-retarded species specific to UAS87. Heat- or proteinase-treatment of the nuclear extract revealed that these species were protein-DNA complexes. The precise binding region of the protein identified by DNaseI protection analysis was found to include a CCAAACA sequence which forms a dyad-symmetrical structure. The amount of one of the three migration-retarded species significantly increased when cells were grown in medium containing a gluconeogenic carbon source. The introduction of pGCR8, a multicopy plasmid containing GCR1 gene, a regulatory gene controlling the expression of several glycolytic enzymes, showed no effect on the amount of three migration-retarded species.     

Occurrence of 9 homologous repeat units in the external spacer region of a nuclear maize rRNA gene unit.

A 14 kb maize DNA fragment carrying nuclear rRNA genes and spacer regions was isolated and characterised by restriction enzyme mapping. A complete 3020 bp long external spacer region was sequenced and revealed 9 tandemly arranged 200 bp long repeat units with high homology. The repeat units lie upstream from two prominent S1 mapping signals. The sequence of a typical repeat unit is compared to a corresponding 130 bp long wheat repeat unit. The possible functional relevance of the repeat units is discussed.     

Characterization of DNA sequences localized 11 to 17.5 kb upstream of the chicken embryonic pi-globin gene

We have analyzed the DNA fragment localized about 11 to 17.5 kb upstream of the chicken alpha-globin gene domain (the fragment was designed as alpha-0). The nucleotide sequence of its 3.3 kb-long 5' part was established and interactions with nuclear matrix proteins were studied. The DNA region localized about 16 kb upstream of the embryonic pi-globin gene showed high affinity to nuclear matrices in vitro. Two palindromes and a cluster of inverted repeats were co-localized in the same region. The whole 6.6 kb alpha-0 fragment decreased the activity of linked CAT reporter gene when transfected into chicken erythroblastoid cells.